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WO2018108165A1 - 一种预防和治疗骨质疏松的药物及其用途 - Google Patents

一种预防和治疗骨质疏松的药物及其用途 Download PDF

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Publication number
WO2018108165A1
WO2018108165A1 PCT/CN2017/116592 CN2017116592W WO2018108165A1 WO 2018108165 A1 WO2018108165 A1 WO 2018108165A1 CN 2017116592 W CN2017116592 W CN 2017116592W WO 2018108165 A1 WO2018108165 A1 WO 2018108165A1
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plasminogen
osteoporosis
mice
bone
subject
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PCT/CN2017/116592
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English (en)
French (fr)
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李季南
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深圳瑞健生命科学研究院有限公司
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Priority to US16/470,167 priority Critical patent/US20190365871A1/en
Priority to EP17880226.0A priority patent/EP3556381A4/en
Priority to JP2019531386A priority patent/JP7531080B2/ja
Priority to CA3047298A priority patent/CA3047298C/en
Priority to CN201780078120.5A priority patent/CN110402150A/zh
Publication of WO2018108165A1 publication Critical patent/WO2018108165A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention relates to the use of plasminogen for the prevention or treatment of osteoporosis and related diseases.
  • Osteoporosis is a systemic disease characterized by reduced bone mass and microstructural destruction of bone tissue, which can lead to increased bone fragility and easy fracture.
  • NASH National Institutes of Health
  • osteoporosis is a skeletal disease characterized by decreased bone strength and increased risk of fracture. Bone strength reflects two major aspects of bone, bone density and bone mass. Osteoporosis leads to a decrease in bone mass and degeneration of bone microstructure, which increases the fragility of the patient's bones and severely reduces the motor function and quality of life of the patient.
  • Mammalian bone development is a highly ordered process that is regulated by multiple factors.
  • the skeletal development of mammals is mainly accomplished by intra-membranous osteogenesis and endochondral ossification.
  • the long bones such as limbs and vertebrae are mainly formed by intra-chondral osteogenesis, and the flat bones such as the skull and the medial part of the clavicle are formed through the membrane.
  • Bone [1] After the formation of bone tissue is not static, but in the steady state dynamic balance of bone formation and absorption. In this process of homeostasis, hormones, multiple signaling pathways, coordinated regulation of bone tissue cells, and homeostasis of mineral salts play an important role [2] .
  • Osteoporosis can be roughly divided into primary and secondary categories, and postmenopausal osteoporosis and senile osteoporosis are primary osteoporosis, and are very common. Secondary osteoporosis is a common systemic bone disease. In addition to known diseases and drugs that induce osteoporosis, some emerging drugs and treatments have become secondary osteoporosis. Important cause.
  • cardiovascular disease A number of studies in recent years have shown that there is a correlation between cardiovascular disease and osteoporosis. They occur together in the elderly, often in the same elderly individual, and the incidence increases with age. Although aging is a common risk factor for cardiovascular disease and osteoporosis, most studies have found that there is still a significant link between the two diseases after ignoring age. On the one hand, cardiovascular disease is associated with loss of bone mass and increased risk of fracture. Similarly, there is evidence that a decrease in bone density can lead to an increase in the incidence and mortality of cardiovascular diseases. Further research found that cardiovascular disease and osteoporosis have a close and direct relationship at the pathogenesis level. Atherosclerosis is the main pathological basis of cardiovascular and cerebrovascular diseases, and arterial calcification is one of its main manifestations.
  • Arterial calcification is considered to be an important marker and clinical monitoring indicator for cardiovascular disease.
  • aortic calcification score (acs) of each patient ranged from 0 to 24 points, aortic calcification was 0, and the most severe aortic calcification was 24 points.
  • the patients were divided into groups according to acs. 3. Bone density was measured using a dual energy x-ray absorptiometer (dxa).
  • Osteoporosis is defined as a bone mineral density value determined by dxa that is less than 2.5 standard deviations or more of the bone peak of the same-sex normal adult. 4.
  • the multivariate regression risk model was used to assess the relationship between aortic calcification and the risk of osteoporosis. At the same time, the study also includes: 1. Screening for eligible postmenopausal women over the age of 60. 2. Semi-quantitative measurement of the degree of aortic calcification. 3. Diagnosis of vertebral fractures: The vertebral fractures were observed by x-ray films (thoracic 4 - lumbar 5 segments) to determine the occurrence of vertebral fractures (the vertebral height decreased by more than 20%). 4.
  • a multivariate regression analysis model was used to assess the relationship between aortic calcification and vertebral fractures.
  • the study also included 1. Screening eligible patients who were over 60 years old were selected as subjects. 2. Bone density measurement and diagnosis of osteoporosis. 3. Carotid and coronary atherosclerotic calcification plaque detection: 64-slice spiral ct carotid artery and coronary cta were used. The 3D image analysis workstation evaluates all cta images. The composition and extent of arterial plaque were evaluated. 4. The multivariate regression risk model was used to analyze the relationship between osteoporosis and bone loss and the risk of carotid and coronary calcification.
  • Osteoporosis is one of the representative symptoms of aging-related conditions and is particularly prevalent in the middle-aged and elderly population. Osteoporosis is a special manifestation of bone aging in biological aging. It has been shown that vitamin D levels are too low or too high to be associated with osteoporosis [11] . With the advent of an aging society, the incidence of osteoporosis is increasing year by year, and the social and economic burden brought about by this is also greatly increased.
  • the bone resorption inhibitor is a drug mainly targeting osteoclasts, which reduces bone resorption by inhibiting the activity of osteoclasts
  • the bone formation promoting agent is a drug mainly for osteoblasts, and can enhance the activity of osteoblasts, Promotes the synthesis of new bone
  • bone mineralization is the basic treatment for osteoporosis, including calcium and vitamin D, which can supplement the bone matrix components.
  • bone resorption inhibitors such as estrogen, bisphosphonates, calcitonin, etc.
  • bone formation enhancers such as parathyroid hormone
  • the invention relates to:
  • WHAT IS CLAIMED IS 1. A method of preventing and treating osteoporosis and related disorders comprising administering to a subject a therapeutically effective amount of plasminogen.
  • osteoporosis comprises primary osteoporosis and secondary osteoporosis.
  • the secondary osteoporosis comprises glucocorticoids, primary hyperparathyroidism, hyperthyroidism, primary biliary cirrhosis, hypogonadism, diabetes, hypertension Osteoporosis caused by atherosclerosis, chronic kidney disease, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, osteoarthritis, gonadal hormone therapy, antiepileptic drug therapy, and chemotherapy drug therapy.
  • a method for preventing and treating osteoporosis complicated by a disease comprising administering to a subject an effective amount of plasminogen, wherein the disease complicated by osteoporosis comprises a glucocorticoid, a primary parathyroid gland Hyperfunction, hyperthyroidism, primary biliary cirrhosis, gonadal function Reduction, diabetes, hypertension, atherosclerosis, chronic kidney disease, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, osteoarthritis, gonadal hormone therapy, antiepileptic drug therapy, chemotherapy drug treatment Loose and loose.
  • the disease complicated by osteoporosis comprises a glucocorticoid, a primary parathyroid gland Hyperfunction, hyperthyroidism, primary biliary cirrhosis, gonadal function Reduction, diabetes, hypertension, atherosclerosis, chronic kidney disease, rheumatoid arthritis, systemic lup
  • a method of preventing osteoporotic fracture comprising administering a subject susceptible to osteoporosis, a subject at high risk for osteoporosis or a subject diagnosed with osteoporosis Plasminogen prevents the occurrence of fractures.
  • the subject comprises a glucocorticoid, a primary hyperparathyroidism, hyperthyroidism, primary biliary cirrhosis, hypogonadism, diabetes, hypertension, Subjects with atherosclerosis, chronic kidney disease, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis or osteoarthritis.
  • a method of enhancing osteoblast activity comprising administering to a subject an effective amount of plasminogen.
  • a method of modulating bone mineral metabolism comprising administering to a subject an effective amount of plasminogen.
  • the regulating comprises lowering blood calcium levels, increasing blood phosphorus levels, promoting deposition of calcium in the bone matrix, and/or reducing deposition of calcium in the vessel wall and viscera.
  • plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, and delta-fibrinolysis Proenzymes or their variants that retain plasminogen activity.
  • plasminogen is a natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • plasminogen is a human plasminogen ortholog from a primate or a rodent or a variant thereof that still retains plasminogen activity Or a fragment.
  • a plasminogen for use in the method of any of items 1-23.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and plasminogen for use in the method of any of items 1-23.
  • a prophylactic or therapeutic kit comprising: (i) plasminogen for use in the method of any of items 1-23 and (ii) for delivery of said plasminogen to The subject of the subject.
  • a plasminogen or a pharmaceutical composition comprising plasminogen comprising the method of any one of clauses 1 to 23, wherein the label indicates administration of the plasminogen or composition
  • the subject is the method of any of clauses 1-23.
  • kit or article of item 30, wherein the other drug comprises a drug that treats osteoporosis or a drug that treats other diseases associated with osteoporosis.
  • An agent for treating osteoporosis comprising plasminogen.
  • a pharmaceutical composition, kit, or article for treating osteoporosis comprising plasminogen.
  • the invention also relates to the use of plasminogen in the manufacture of a medicament, a pharmaceutical composition, an article, a kit for use in the method of any of the above items 1-23.
  • the plasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97 with sequence 2, 6, 8, 10 or 12. %, 98% or 99% sequence identity and still have plasminogen activity.
  • the plasminogen is added, deleted, and/or substituted on the basis of sequence 2, 6, 8, 10, or 12, 1-100, 1-90, 1-80, 1-70 , 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1 -3, 1-2, 1 amino acid, and still a protein with plasminogen activity.
  • the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, delta-plasminogen, or their retention A variant of plasminogen activity.
  • the plasminogen is a native or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • the plasminogen is a human plasminogen ortholog from a primate or a rodent or a variant or fragment thereof that still retains plasminogen activity.
  • the amino acid of plasminogen is as shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is human natural plasminogen.
  • the subject is a human. In some embodiments, the subject lacks or lacks plasminogen. In some embodiments, the deficiency or deficiency is innate, secondary, and/or local.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and plasminogen for use in the foregoing methods.
  • the kit can be a prophylactic or therapeutic kit comprising: (i) plasminogen for use in the foregoing methods and (ii) for delivery of the fibrinolysis The zymogen is to the subject of the subject.
  • the member is a syringe or vial.
  • the kit further comprises a label or instructions for use, the label or instructions for use indicating administration of the plasminogen to the subject to perform any of the methods described above.
  • the article of manufacture comprises: a container comprising a label; and a pharmaceutical composition comprising (i) plasminogen or a plasminogen for use in the foregoing method, wherein the label indicates that the fiber is to be The lysogen or composition is administered to the subject to perform any of the methods described above.
  • the kit or article further comprises an additional one or more components or containers containing other drugs.
  • the other drug is selected from the group consisting of a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, a hepatoprotective drug, and an anti-heart rhythm Abnormal drugs, cardiotonic drugs, diuretic drugs, anti-infective drugs, antiviral drugs, immunomodulatory drugs, inflammatory regulatory drugs and anti-tumor drugs.
  • the plasminogen is administered systemically or locally, preferably by the following route: intravenous, intramuscular, subcutaneous administration of plasminogen for treatment.
  • the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer.
  • the plasminogen is 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg per day.
  • the present invention expressly covers all combinations of the technical features between the embodiments of the present invention, and these combined technical solutions are explicitly disclosed in the present application, just as the above technical solutions have been separately and explicitly disclosed.
  • the present invention also expressly encompasses all subcombinations of the various embodiments and elements thereof, and is disclosed herein as if each such subcombination is separately and explicitly disclosed herein.
  • Ostoporosis is a systemic degenerative bone disease characterized by low bone mass, damaged bone microstructure, increased bone fragility and prone to fracture. Generally divided into three categories of primary, secondary and idiopathic osteoporosis.
  • Primary osteoporosis is divided into postmenopausal osteoporosis (type I) and senile osteoporosis (type II), in which postmenopausal osteoporosis generally occurs within 5 to 10 years after menopause Osteoporosis is generally referred to as osteoporosis in the elderly aged 60 years and older.
  • Primary osteoporosis mainly emphasizes the important role of bone mass, bone loss and bone structure. It is characterized by reduced bone mass, increased fragility, structural deterioration and prone to fracture.
  • Bone osteoporosis refers to a disease in which the bone structure is reduced due to certain diseases, drugs or other causes, and the microstructure of the bone changes, which is prone to fragility fracture. Common diseases or drugs that cause osteoporosis include:
  • glucocorticoids Excessive glucocorticoids, excessive replacement of thyroid hormones, antiepileptic drugs, lithium or aluminum poisoning, cytotoxic or immunosuppressive agents (cyclosporine A, tacrolimus), heparin, drugs that cause hypogonadism (aromatase inhibitors) Gonadotropin-releasing hormone analogues, etc.)
  • Chronic liver disease especially primary biliary cirrhosis
  • inflammatory bowel disease especially Crohn's disease
  • major gastric resection diarrhea
  • Osteogenesis imperfecta Marfan syndrome, hemochromatosis, homocystinuria, porphyria
  • osteoporosis causes osteoporosis by affecting osteoblast and osteoclast function, resulting in increased bone resorption and/or reduced bone formation.
  • second osteoporosis encompasses osteoporosis caused by various reasons as described above.
  • Idiopathic osteoporosis mainly occurs in adolescents. It generally refers to osteoporosis in which the age of onset of men is less than 50 years and the age of onset of women is less than 40 years old. There is no underlying disease, and the cause is unknown.
  • Concurrent osteoporosis with a disease or condition refers to osteoporosis that occurs with the disease or condition. There may be some inherent etiology or pathogenesis association between the disease or condition and osteoporosis. For example, osteoporosis complicated by diabetes, osteoporosis complicated by atherosclerosis, osteoporosis complicated by chronic kidney disease, osteoporosis complicated by ankylosing spondylitis, and osteoporosis complicated by osteoarthritis.
  • Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease that hydrolyzes several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans [12] . In addition, plasmin activates some metalloproteinase precursors (pro-MMPs) to form active metalloproteinases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis [13-14] . Plasmin is formed by proteolytic plasminogen by two physiological PAs: tissue plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA).
  • tPA tissue plasminogen activator
  • uPA urokinase-type plasminogen activator
  • PAI-1 plasminogen activator inhibitor-1
  • PAI-2 lysogen activator inhibitor-2
  • Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa [17-18] . Plasminogen is mainly synthesized in the liver and is abundantly present in the extracellular fluid. The plasma has a plasminogen content of about 2 ⁇ M. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids [19-20] . Plasminogen exists in two molecular forms: glutamate-plasminogen and Lys-plasminogen. The naturally secreted and uncleaved forms of plasminogen have an amino terminal (N-terminal) glutamate and are therefore referred to as glutamate-plasminogen.
  • plasminogen glutamate-plasminogen is hydrolyzed to Lys-Lysinogen at Lys76-Lys77.
  • lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate.
  • the Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of a disulfide-linked double-chain protease plasmin [21] .
  • the amino terminal portion of plasminogen contains five homologous tricycles, the so-called kringles, which contain a protease domain.
  • Some kringles contain a lysine binding site that mediates the specific interaction of plasminogen with fibrin and its inhibitor alpha2-AP.
  • the main substrate for plasmin is fibrin, which is the key to preventing pathological thrombosis [22] .
  • Plasmin also has substrate specificity for several components of ECM, including laminin, fibronectin, proteoglycans and gelatin, suggesting that plasmin also plays an important role in ECM reconstruction [18,23- 24] .
  • plasmin can also degrade other components of ECM, including MMP-1, MMP-2, MMP-3 and MMP-9, by converting certain protease precursors into active proteases. Therefore, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis [25] .
  • plasmin has the ability to activate certain potential forms of growth factors [26-28] . In vitro, plasmin also hydrolyzes components of the complement system and releases chemotactic complement fragments.
  • Plasmid is a very important enzyme found in the blood that hydrolyzes fibrin clots into fibrin degradation products and D-dimers.
  • Plasinogen is a zymogen form of plasmin, which is composed of 810 amino acids, based on the sequence in swiss prot, based on the native human plasminogen amino acid sequence (sequence 4) containing the signal peptide. 90 kD, a glycoprotein synthesized mainly in the liver and capable of circulating in the blood, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO:3.
  • Full-length plasminogen contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle 1-5).
  • the serine protease domain includes the residues Val581-Arg804.
  • Glu-plasminogen is a natural full-length plasminogen consisting of 791 amino acids (not containing a 19 amino acid signal peptide), and the cDNA sequence encoding the sequence is shown in SEQ ID NO: 1, and its amino acid sequence is sequence 2. Shown. In vivo, there is also a Lys-plasminogen which is hydrolyzed from amino acids 76-77 of Glu-plasminogen, and as shown in SEQ ID NO: 6, the cDNA sequence encoding the amino acid sequence is as shown in SEQ ID NO: 5 Shown.
  • Delta-plasminogen is a fragment of full-length plasminogen lacking the Kringle2-Kringle5 structure, containing only Kringle1 and serine protease domains [29-30] , and has been reported in the literature for delta-plasminogen.
  • the amino acid sequence (SEQ ID NO: 8) [31] the cDNA sequence encoding the amino acid sequence is shown in Sequence 7.
  • Mini-plasminogen consists of Kringle5 and a serine protease domain, which has been reported in the literature to include the residue Val443-Asn791 (starting amino acid with a Glu residue of Glu-plasminogen sequence not containing a signal peptide) [31] , the amino acid sequence thereof is shown in SEQ ID NO: 10, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 9.
  • Micro-plasminogen contains only the serine protease domain, and its amino acid sequence has been reported to include the residue Ala543-Asn791 (from the Glu residue of the Glu-plasminogen sequence containing no signal peptide).
  • Plasin of the present invention is used interchangeably with “fibrinolytic enzyme” and “fibrinolytic enzyme”, and has the same meaning; “plasminogen” and “plasminogen”, “fibrinogenase” "Interchangeable use, meaning the same.
  • the meaning or activity of the "deficiency" of plasminogen is such that the level of plasminogen in the subject is lower than that of a normal person, which is low enough to affect the normal physiological function of the subject;
  • the meaning or activity of plasminogen "deletion” is that the level of plasminogen in the subject is significantly lower than that of normal people, and even the activity or expression is minimal, and only by external supply can maintain normal physiological function.
  • plasminogen adopts a closed inactive conformation, but when bound to the surface of a thrombus or cell, it is converted to openness mediated by plasminogen activator (PA).
  • PA plasminogen activator
  • Conformational active plasmin The active plasmin further hydrolyzes the fibrin clot into a fibrin degradation product and a D-dimer, thereby dissolving the thrombus.
  • the PAp domain of plasminogen contains an important determinant that maintains plasminogen in an inactive blocking conformation, while the KR domain is capable of binding to lysine residues present on the receptor and substrate.
  • plasminogen activators include tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hag Mann factor) and so on.
  • a "plasminogen active fragment” refers to an active fragment that binds to a target sequence in a substrate and exerts a proteolytic function in a plasminogen protein.
  • the technical solution of the present invention relating to plasminogen covers the technical solution of replacing plasminogen with a plasminogen active fragment.
  • the plasminogen active fragment of the present invention is a protein comprising a serine protease domain of plasminogen.
  • the plasminogen active fragment of the present invention comprises the sequence 14, and the sequence 14 has at least 80%, 90.
  • the plasminogen of the present invention comprises a protein comprising the plasminogen active fragment and still retaining the plasminogen activity.
  • blood plasminogen and its activity assays include: detection of tissue plasminogen activator activity (t-PAA), detection of plasma tissue plasminogen activator antigen (t-PAAg), Detection of plasma tissue plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity, inhibition of plasma tissue plasminogen activator Detection of antigens, plasma plasmin-anti-plasmin complex assay (PAP).
  • t-PAA tissue plasminogen activator activity
  • t-PAAg detection of plasma tissue plasminogen activator antigen
  • plgA Detection of plasma tissue plasminogen activity
  • plgAg detection of plasma tissue plasminogen antigen
  • PAP plasma plasmin-anti-plasmin complex assay
  • the most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and chromogenic substrate to the plasma to be tested, and the PLG in the tested plasma is converted into PLM under the action of SK, and the latter acts on The chromogenic substrate is then measured spectrophotometrically and the increase in absorbance is directly proportional to the plasminogen activity.
  • plasminogen activity in blood can also be measured by immunochemical method, gel electrophoresis, immunoturbidimetry, or radioimmunoassay.
  • Ortholog or ortholog refers to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs, orthologs. It specifically refers to a protein or gene that has evolved from the same ancestral gene in different species.
  • Lysozymes include human natural plasminogen and also include plasminogen orthologs or orthologs of plasminogen activity from different species.
  • Constant substitution variant refers to a change in one of the given amino acid residues without altering the overall conformation and function of the protein or enzyme, including but not limited to similar properties (eg, acidic, basic, hydrophobic, etc.)
  • the amino acid replaces the amino acid in the amino acid sequence of the parent protein.
  • Amino acids having similar properties are well known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and are interchangeable.
  • isoleucine is a hydrophobic amino acid that can be replaced by leucine, methionine or valine. Therefore, the similarity of two protein or amino acid sequences of similar function may be different.
  • Constant substitution variants also includes determining polypeptides or enzymes having more than 60% amino acid identity by BLAST or FASTA algorithm. If it is more than 75%, preferably more than 85%, or even more than 90%. Optimal and have the same or substantially similar properties or functions as the native or parent protein or enzyme.
  • Isolated plasminogen refers to a plasminogen protein that is isolated and/or recovered from its natural environment.
  • the plasminogen will purify (1) to a purity greater than 90%, greater than 95%, or greater than 98% by weight, as determined by the Lowry method, eg, over 99% (by weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequence analyzer, or (3) to homogeneity, which is by use Coomassie blue or silver staining was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions.
  • Isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.
  • polypeptide peptide
  • protein protein
  • fusion proteins including, but not limited to, fusion proteins having a heterologous amino acid sequence, fusions having heterologous and homologous leader sequences (with or without an N-terminal methionine residue);
  • the "percent amino acid sequence identity (%)" with respect to a reference polypeptide sequence is defined as the introduction of a gap as necessary to achieve maximum percent sequence identity, and without any conservative substitution being considered as part of sequence identity, in the candidate sequence
  • the amino acid residues in the reference polypeptide sequence are the same Percentage of amino acid residues. Comparisons for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art will be able to determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum contrast over the full length of the sequences being compared. However, for the purposes of the present invention, amino acid sequence identity percent values are generated using the sequence comparison computer program ALIGN-2.
  • amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or may be expressed as having or comprising relative to, and, or for a given amino acid sequence)
  • a given amino acid sequence A of a certain % amino acid sequence identity of B is calculated as follows:
  • X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 in the A and B alignments of the program, and wherein Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A. All % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph, unless explicitly stated otherwise.
  • the terms “treating” and “treating” refer to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be to completely or partially prevent the disease or its symptoms, and/or to partially or completely cure the disease and/or its symptoms, and includes: (a) preventing the disease from occurring in the subject, the subject may have The cause of the disease, but not yet diagnosed as having a disease; (b) inhibiting the disease, ie, retarding its formation; and (c) reducing the disease and/or its symptoms, ie causing the disease and/or its symptoms to subside.
  • the terms "individual”, “subject” and “patient” are used interchangeably herein to refer to a mammal, including but not limited to a mouse (rat, mouse), a non-human primate, a human, a dog, a cat. Hoofed animals (such as horses, cattle, sheep, pigs, goats).
  • “Therapeutically effective amount” or “effective amount” refers to an amount of plasminogen sufficient to effect such prevention and/or treatment of a disease when administered to a mammal or other subject to treat the disease.
  • the “therapeutically effective amount” will vary depending on the plasminogen used, the severity of the disease and/or its symptoms of the subject to be treated, and the age, weight, and the like.
  • Plasminogen can be isolated and purified from nature for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is chemically synthesized, it can be synthesized in a liquid phase or a solid phase.
  • Solid phase polypeptide synthesis SPPS
  • Fmoc and Boc Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen.
  • the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected to reveal a new N-terminal amine that can be attached to other amino acids.
  • the peptide remains immobilized on the solid phase and then cut off.
  • the plasminogen of the present invention can be produced using standard recombinant methods.
  • a nucleic acid encoding plasminogen is inserted into an expression vector operably linked to a regulatory sequence in an expression vector.
  • Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • Expression regulation can be a eukaryotic promoter system in a vector that is capable of transforming or transfecting eukaryotic host cells (eg, COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of plasminogen.
  • Suitable expression vectors are typically replicated as an episome in the host organism or as an integral part of the host chromosomal DNA.
  • expression vectors typically contain a selection marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate transformation of the desired DNA sequence with foreign sources. Those cells are tested.
  • a selection marker eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance
  • Escherichia coli is an example of a prokaryotic host cell that can be used to clone a subject antibody-encoding polynucleotide.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Genus (Pseudomonas) species.
  • expression vectors can also be generated which will typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell.
  • promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from phage lambda. Promoters typically control expression, optionally in the context of manipulating a gene sequence, and have a ribosome binding site sequence, etc., to initiate and complete transcription and translation.
  • yeast can also be used for expression.
  • Yeast e.g., S. cerevisiae
  • Pichia are examples of suitable yeast host cells in which a suitable vector has expression control sequences (e.g., a promoter), an origin of replication, a termination sequence, and the like, as desired.
  • a typical promoter comprises 3-phosphoglycerate kinase and other saccharolytic enzymes.
  • Inducible yeast is initiated by a promoter specifically comprising an alcohol dehydrogenase, an isocytochrome C, and an enzyme responsible for the utilization of maltose and galactose.
  • mammalian cells e.g., mammalian cells cultured in in vitro cell culture
  • an anti-Tau antibody of the invention e.g., a polynucleotide encoding a subject anti-Tau antibody.
  • Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for these cells may contain expression control sequences such as origins of replication, promoters and enhancers (Queen et al, Immunol. Rev.
  • RNA splice sites sites that are ribosome binding.
  • RNA splice sites sites that are ribosome binding.
  • polyadenylation sites sites that are ribosome binding sites.
  • transcription terminator sequences sites that are ribosome binding sites.
  • suitable expression control sequences are promoters derived from the white immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J. Immunol. 148: 1149 (1992).
  • the invention may be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and the like.
  • Plasminogen is substantially pure, such as at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure. Or more pure, for example, free of contaminants, such as cellular debris, macromolecules other than the subject antibody, and the like.
  • Therapeutic formulations are prepared as a lyophilized formulation or as an aqueous solution.
  • Acceptable carriers, excipients, and stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as Octadecyldimethylbenzylammonium chloride; chlorinated hexane diamine; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoic acid Esters such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptide (less than about 10 residues) Protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, his
  • the formulations of the invention may also contain more than one active compound as required for the particular condition being treated, preferably those having complementary activities and no side effects to each other.
  • active compound for example, antihypertensive drugs, antiarrhythmic drugs, drugs for treating diabetes, and the like.
  • the plasminogen of the present invention may be encapsulated in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, for example, may be placed in a glial drug delivery system (eg, liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules are placed in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion.
  • glial drug delivery system eg, liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules are placed in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion.
  • the plasminogen of the invention for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter before or after lyophilization and reconstitution.
  • the plasminogen of the present invention can prepare a sustained release preparation.
  • sustained release formulations include solid hydrophobic polymeric semi-permeable matrices having a shape and containing glycoproteins, such as films or microcapsules.
  • sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al, J. Biomed. Mater.
  • Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can sustain release molecules for more than 100 days, while some hydrogels release proteins for shorter periods of time.
  • a rational strategy for stabilizing proteins can be designed based on relevant mechanisms. For example, if the mechanism of aggregation is found to be an intermolecular SS bond by thiodisulfide bond exchange, it can be modified by modifying the thiol residue, lyophilizing from an acidic solution, controlling humidity, using suitable additives, and developing specific The polymer matrix composition is used to achieve stability.
  • compositions of this invention may be effected intramuscularly in different ways, such as by intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery).
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffering media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils.
  • Intravenous vehicles contain liquid and nutritional supplements, electrolyte supplements, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like.
  • the medical staff will determine the dosage regimen based on various clinical factors. As is well known in the medical arts, the dosage of any patient depends on a variety of factors, including the patient's size, body surface area, age, specific compound to be administered, sex, number and route of administration, overall health, and other medications administered simultaneously. .
  • the pharmaceutical composition of the present invention comprising plasminogen may have a dose ranging, for example, from about 0.0001 to 2000 mg/kg per day, or from about 0.001 to 500 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75). Mg/kg, 10 mg/kg, 50 mg/kg, etc.) Subject weight.
  • the dose can be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1-50 mg/kg, or at least 1 mg/kg. Dosages above or below this exemplary range are also contemplated, particularly in view of the above factors. Intermediate doses in the above ranges are also included in the scope of the present invention.
  • the subject can administer such doses daily, every other day, every week, or according to any other schedule determined by empirical analysis.
  • An exemplary dosage schedule includes 1-10 mg/kg for several days. The therapeutic effect and safety need to be evaluated in real time during the administration of the drug of the present invention.
  • One embodiment of the invention relates to an article or kit comprising a plasminogen or plasmin of the invention useful for the treatment of osteoporosis and related conditions.
  • the article preferably includes a container, label or package insert. Suitable containers are bottles, vials, syringes, and the like.
  • the container can be made of various materials such as glass or plastic.
  • the container contains a composition that is effective to treat a disease or condition of the invention and has a sterile access port (eg, the container can be an intravenous solution or vial containing a stopper that can be penetrated by a hypodermic needle) of). At least one active agent in the composition is plasminogen/plasmin.
  • the label on or attached to the container indicates that the composition is used to treat the osteoporosis and related conditions of the present invention.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • the article comprises a package insert with instructions for use, including, for example, a user instructing the composition to administer the plasminogen composition and other drugs to treat the accompanying disease.
  • One embodiment of the invention relates to the determination of therapeutic efficacy and therapeutic safety following treatment of a subject with plasminogen.
  • Commonly used osteoporosis treatment monitoring and evaluation includes follow-up (adverse reactions, standardized medication, basic measures and re-evaluation of fracture risk factors), new fracture assessment (clinical fracture, height reduction and imaging examination), bone mineral density (bone mineral density, BMD) measurement and bone turnover markers (BTM) detection, and comprehensive reassessment based on these data.
  • BMD and bone mass are the most widely used methods for monitoring and evaluating therapeutic effects. For example, dual energy X-ray absorptiometry (DXA), quantitative computed tomography (QCT), single photon can be used.
  • DXA dual energy X-ray absorptiometry
  • QCT quantitative computed tomography
  • BMD Absorption assay
  • SPA Absorption assay
  • BMD can be detected once a year after the start of treatment, and the interval can be appropriately extended after the BMD has stabilized, for example, once every 2 years.
  • the bone formation index currently used in serological indicators is procollagen type 1n-terminal propeptide (PINP), and the bone resorption index is serum type 1 procollagen C-terminal peptide ( Serum C-terminal telopeptide, S-CTX).
  • PINP procollagen type 1n-terminal propeptide
  • S-CTX serum type 1 procollagen C-terminal peptide
  • Baseline values should be measured prior to initiation of treatment, 3 months after application of drug-promoting therapy, and 3 to 6 months after application of inhibitory drug therapy.
  • BTM can provide dynamic information of bones, independent of BMD in function and function, and also complements BMD as a monitoring tool.
  • the combination of the two has higher clinical value.
  • BMD rises or stabilizes after treatment BTM has an expected change, and no fracture occurs during treatment, the treatment response is considered to be good.
  • the present invention relates to the use of plasminogen and variants thereof for the treatment of a subject during and after treatment, the determination of the safety of the treatment regimen, including but not limited to serum for the drug in the subject Half-life, treatment half-life, half-toxicity (TD50), and median lethal dose (LD50) were counted, or various adverse events such as sensitization reactions occurred during or after treatment were observed.
  • TD50 treatment half-life
  • LD50 median lethal dose
  • Figure 1 shows representative pictures of knee joint Safranin O staining of 15-week old wild-type and plasminogen-deficient mice.
  • A is a wild type mouse and B is a plasminogen deficient mouse. Plasminogen-deficient mice exhibited extensive osteopenia and increased bone marrow cells compared to wild-type mice.
  • FIG. 2 shows the results of blood calcium detection in 15 weeks old plasminogen deficient and wild type mice.
  • the results showed that the serum calcium levels of plasminogen-deficient (Ko) mice were significantly higher than those of wild-type mice (Wt), and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen plays an important role in maintaining normal calcium metabolism.
  • Figure 3 shows the results of H&E staining of knee joints after 30 days of administration of plasminogen by plasminogen-deficient (Plg -/- ) mice.
  • a and C were given to the vehicle PBS control group, and B and D were given to the plasminogen group.
  • the results showed that the growth plate (arrow mark) was disordered in the vehicle PBS control group, and the bone marrow disappeared in some bone marrow cavity (triangular mark); the plasminogen group and the growth plate (arrow mark) were arranged neatly. This indicates that plasminogen can promote the normal growth of knee growth plates in Plg -/- mice.
  • Figure 4 shows the results of alkaline phosphatase staining of knee joint articular cartilage 30 days after administration of plasminogen by Plg -/- mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that only a small amount of alkaline phosphatase was stained on the surface of the articular cartilage in the vehicle PBS control group, while the surface of the articular cartilage in the plasminogen group was more colored with dark red alkaline phosphatase (arrow mark).
  • the alkaline phosphatase activity on the surface of articular cartilage in the plasminogen group was significantly higher than that in the PBS control group, that is, plasminogen significantly increased the osteoblast activity of the articular cartilage of the knee joint.
  • Figure 5 shows the results of alkaline phosphatase staining of knee growth plates after 30 days of administration of plasminogen by Plg -/- mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that the vehicle PBS control group had alkaline phosphatase staining (arrow mark) at the activity of growth plate osteoblasts, which was light red; the plasminogen group had more alkaline phosphatase staining at the growth plate. It is dark red. After the administration of plasminogen, it can promote the increase of osteoblast activity of the knee growth plate.
  • Figure 6 shows the results of serum alkaline phosphatase assay after 28 days of administration of plasminogen in 0.5 ⁇ g/kg vitamin D aging model C57 mice.
  • the results showed that serum alkaline phosphatase activity in the plasminogen group was significantly higher than that in the control vehicle PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05), and compared with the vehicle control PBS control group.
  • the serum alkaline phosphatase activity of the plasminogen group mice was closer to that of the blank control group. This suggests that the plasminogen group can significantly promote the increase in osteoblast activity in the vitamin D aging model mice.
  • Figure 7 shows a representative picture of alkaline phosphatase staining of a lycopene 28 day knee joint growth plate in a 1 ⁇ g/kg vitamin D aging model C57 mouse.
  • A is a blank control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results showed that the alkaline phosphatase positive staining (arrow mark) of the knee growth plate in the vehicle PBS control group was significantly less than that of the blank control mice; the alkaline phosphatase positive staining of the knee growth plate in the plasminogen group was significantly higher than that given to the plasminogen group.
  • Solvent PBS control mice This indicates that plasminogen can improve the activity of osteoblasts in the knee joint growth plate of vitamin D-induced aging model mice.
  • FIG. 8 Results of micro-CT bone density measurements of the skulls of Plg -/- and Plg +/+ mice at different ages.
  • A is the cortical bone density
  • B is the total bone density of the skull.
  • the results showed that with the increase of age, the bone density and total bone density of Plg +/+ mice gradually increased, while the bone density and total bone density of the skull bone of Plg -/- mice gradually decreased.
  • the bone density of the two strains was extremely significant at 20-21 weeks of age, and the difference was more pronounced at 29-30 weeks of age. This indicates that plasminogen plays an important role in the regulation of bone mineral density in the cranium and is closely related to osteoporosis.
  • FIG. 9 Results of bone mineral content of the skull bone micro CT of Plg /- and Plg +/+ mice at 20-21 weeks of age. The results showed that the mineral content of cortical bone and total bone in Plg +/+ mice at 20-21 weeks old was significantly higher than that in Plg /- mice, and the statistical difference was significant. It indicates that plasminogen plays an important role in the regulation of mineral content of cranium and is closely related to osteoporosis.
  • FIG. 10 Results of bone microscopy of femur Micro CT in Plg -/- and Plg +/+ mice at different ages.
  • A cortical bone density
  • B cancellous bone density
  • C trabecular bone density
  • D total bone density.
  • the results showed that the femur density of Plg +/+ mice increased gradually with the increase of age during the period of 12-30 weeks, while the bone density, cancellous bone density, trabecular bone density and total of femur cortex in Plg -/- mice The bone density is gradually reduced.
  • FIG. 11 Results of bone mineral content of femur Micro CT in different age groups of Plg -/- and Plg +/+ mice.
  • A is the mineral content of cortical bone
  • B is the mineral content of cancellous bone
  • C is the mineral content of trabecular bone
  • D is the total bone mineral content.
  • the results showed that mineral content in different parts of the femur of Plg +/+ mice did not change or gradually increased with the increase of age during the period of 12-30 weeks, while the Feg -/- mice femoral cancellous bone and trabecular bone minerals The content gradually decreases.
  • Figure 12 Results of micro-CT bone density measurements of lumbar vertebrae in Plg -/- and Plg +/+ mice at different ages.
  • A cortical bone density
  • B cancellous bone density
  • C trabecular bone density
  • D total bone density.
  • the results showed that the bone mineral density of lumbar vertebrae in Plg +/+ mice increased gradually with the age of 12-30 weeks, while the bone density, cancellous bone density, trabecular bone of lumbar vertebrae in Plg -/- mice Bone mineral density and total bone density gradually decrease. During this period, the bone density of Plg +/+ mice was higher than that of Plg -/- mice.
  • the bone mineral density of lumbar vertebrae in the two strains was significantly different, and the difference was observed with the increase of age. More and more significant. It indicates that plasminogen is involved in the regulation of lumbar spine bone density and plays an important role in a certain period of time.
  • Fig. 13 Results of bone mineral content of lumbar vertebrae Micro CT in different age groups of Plg -/- and Plg +/+ mice.
  • A is the mineral content of cortical bone
  • B is the mineral content of cancellous bone
  • C is the mineral content of trabecular bone
  • D is the total bone mineral content.
  • the results showed that the mineral content of different parts of lumbar vertebrae of Plg +/+ mice did not change much with the increase of age during the period of 12-30 weeks, while the lumbar vertebrae, cancellous bone and trabecular bone of Plg -/- mice The bone and total bone mineral content gradually decreased.
  • Figure 14 shows the results of serum alkaline phosphatase assay in Plg -/- and Plg +/+ mice at different ages.
  • the results showed that the serum alkaline phosphatase activity of Plg +/+ mice did not change significantly at 12-30 weeks of age, but the change of serum alkaline phosphatase activity in Plg -/- mice was related to the age of the week.
  • the gradual decrease was increased; the activity of serum alkaline phosphatase in Plg +/+ mice was significantly higher than that in Plg -/- mice, and the serum alkaline phosphatase activity of the two strains showed significant difference at 12 weeks of age. And as the age of the week increases, the difference becomes more and more significant. This result suggests that plasminogen may promote osteoblast activity and promote bone remodeling.
  • Figure 15 shows the results of blood calcium test after 30 days of administration of plasminogen in ApoE atherosclerotic model mice. The results showed that the serum calcium concentration in the plasminogen group was significantly lower than that in the vehicle control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can reduce the serum calcium content of ApoE atherosclerotic model mice.
  • Figure 16 shows a representative picture of aortic sinusin red staining after 30 days of administration of plasminogen in ApoE atherosclerotic model mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that the calcium deposition in the aortic sinus of the plasminogen group was significantly less than that in the vehicle PBS control group. This indicates that plasminogen can improve aortic sinus calcification in atherosclerosis.
  • FIG. 17 Results of femur density after administration of plasminogen in atherosclerotic model mice.
  • A cortical bone density
  • B cancellous bone density
  • C trabecular bone density
  • D total bone density.
  • the results showed that the femoral density of the plasminogen group was significantly higher than that of the vehicle control group after 10 days of administration, and the difference in cancellous bone density and total bone density was significant (* indicates P ⁇ 0.05);
  • the cancellous bone mineral density, trabecular bone density and total bone density of the plasminogen group were significantly higher than those of the vehicle control group (P ⁇ 0.05).
  • FIG. 18 Representative pictures of knee joint H&E staining after administration of plasminogen in atherosclerotic model mice.
  • A-C was the vehicle PBS control group and D-F was the plasminogen group.
  • the results showed that the cartilage surface of the vehicle PBS control group was mildly fibrillated (thin arrow mark), the trabecular bone (triangle mark) was thinned, the thickness was uneven, the cartilage tissue (star mark) was disorderly arranged, and the growth plate (thick arrow) Marking)
  • the level is disordered, the chondrocytes are slightly reduced, the tidal line is basically clear; the surface of the articular cartilage in the plasminogen group is basically normal, the tidal line is clear, the thickness of the trabecular bone is uniform, the structure of the growth plate is clear, and the hierarchy is regular and separable. This indicates that plasminogen can improve the condition of knee joint in ApoE atherosclerotic model mice.
  • Figure 19 shows the effect of plasminogen on body weight of C57 ovariectomized and dexamethasone-induced osteoporosis model mice.
  • the results showed that the body weight of the vehicle control group was significantly lighter than that of the normal control group, while the body weight of the plasminogen group was significantly higher than that of the vehicle control group, and the statistical difference was significant. (P ⁇ 0.05). This indicates that plasminogen can significantly promote ovarian resection and injection of dexamethasone-induced osteoporosis in mice.
  • Figure 20 shows the results of femur micro CT scan of C57 ovariectomy and dexamethasone-induced osteoporosis model mice after administration of plasminogen.
  • A is the bone volume measurement result
  • B is the bone mineral content measurement result.
  • the results showed that the cancellous bone, trabecular bone, total bone volume and bone mineral content of the femur in the plasminogen group were larger than those in the vehicle control group, and the statistical differences were significant (* indicates P ⁇ 0.05). This indicates that plasminogen can promote the deposition of femur minerals, increase bone mass and improve osteoporosis in osteoporosis model mice.
  • Figure 21 shows the results of femur micro CT scan of plasminogen C57 ovariectomy and injection of dexamethasone-induced osteoporosis model mice.
  • Figure 21A shows the femur bone density measurement results.
  • the results showed that the femoral cortical bone, cancellous bone, trabecular bone and total bone bone density of the vehicle in the PBS control group were lower than those in the normal control group; while the bone density of each part in the plasminogen group was greater than that of the vehicle PBS control. group.
  • the trend is clear, but due to the small number of mice, the statistical difference is only close to significant. It can be expected that there will be statistical differences in increasing the number of mice.
  • Figure 21B shows the measurement of mineral content of the femur bone.
  • the results showed that the bone mineral content of the femur in the PBS control group was lower than that in the normal control group, while the bone mineral content in the plasminogen group was higher than that in the vehicle PBS control group.
  • the trend is clear, but due to the small number of mice, the statistical difference is only close to significant. It can be expected that there will be statistical differences in increasing the number of mice.
  • Figure 21C shows the trabecular bone volume measurement results.
  • the results showed that the trabecular bone volume of the femur in the control vehicle PBS control group was smaller than that in the normal control group, while the femoral trabecular bone volume in the plasminogen group was greater than that in the vehicle PBS control group.
  • the trend is clear, but due to the small number of mice, the statistical difference is only close to significant. It can be expected that there will be statistical differences in increasing the number of mice.
  • plasminogen can significantly improve osteoporosis, promote bone mineral density and bone mass increase in various parts of the femur, and the improvement of trabecular bone is particularly obvious.
  • Figure 22 is a representative picture of knee joint H&E staining and Safrain O staining of plasminogen C57 ovariectomy and injection of dexamethasone-induced osteoporosis model mice.
  • a and C are for the vehicle PBS group
  • B and D are for the plasminogen group.
  • the results showed that the trabecular bone (arrow mark) of the PBS group was significantly thinner and fractured, and a large area of the trabecular bone marrow cavity was formed. The medullary cavity was enlarged, the connection of the trabecular bone was interrupted, and the growth of the bone cells under the plate was light.
  • Figure 23 is a representative picture of alkaline phosphatase staining of knee joints in mice with osteoporosis induced by plasminogen C57 ovariectomy and injection of dexamethasone.
  • a and C were given to the vehicle PBS control group, and B and D were given to the plasminogen group.
  • the results showed that the alkaline phosphatase staining of the knee joint cartilage tissue (thin arrow mark) and the growth plate (thick arrow mark) of the vehicle PBS control group was significantly less than that of the plasminogen group. This indicates that plasminogen promotes the increase of knee osteoblast activity in osteoporosis model mice.
  • Figure 24 shows serum calcium test results in osteoporosis model mice induced by Plg + / + ovariectomy after administration of plasminogen.
  • the results showed that the serum calcium concentration in the plasminogen group was significantly lower than that in the vehicle PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can significantly reduce the concentration of serum calcium in ovariectomized osteoporosis model mice.
  • Figure 25 shows the results of serum phosphorus detection in osteoporosis model mice induced by Plg +/+ ovariectomy after administration of plasminogen.
  • the results showed that the serum phosphorus concentration in the plasminogen group was significantly higher than that in the vehicle PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can significantly increase the blood phosphorus concentration in ovariectomized osteoporosis model mice.
  • Figure 26 shows the results of alkaline phosphatase staining of knee joints in mice with plasminogen 3% cholesterol hyperlipidemia.
  • a and C are the control group for the vehicle PBS
  • B and D are for the plasminogen group
  • E is the quantitative analysis result.
  • the results showed that the alkaline phosphatase coloration (arrow mark) of the knee joint of the plasminogen group was significantly more than that of the vehicle control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen significantly increases knee osteoblast activity in 3% cholesterol hyperlipidemia model mice.
  • Figure 27 is a representative picture of H&E staining and Safrain O staining of knee joints in mice with osteoporosis induced by plasminogen C57 ovariectomy and injection of dexamethasone.
  • a and C are for the vehicle PBS group
  • B and D are for the plasminogen group.
  • the results showed that the skeletal trabecular bone (arrow mark) of the control group of the vehicle was significantly thinned and broken, and a large area of the trabecular bone marrow cavity was found. The connection of the trabecular bone was interrupted, and the surface of the joint was partially fibrillated.
  • the osteogenesis of the osteogenesis area under the growth plate was significantly reduced (triangular mark); the trabecular bone part of the plasminogen group was thinned, and the trabecular bone was better than the PBS control group, and there was no serious fracture. Without a large area of the trabecular trabecular region, the cartilage tissue hierarchy is more regular and the tidal line is clear. This indicates that administration of plasminogen can significantly improve the condition of the knee joint in osteoporosis model mice.
  • mice C57 mice and Plg +/+ and Plg ⁇ / ⁇ mice (Jackson Lab) were used for related experiments. Animals are kept in an environment in which experimental animals are used in accordance with national standards.
  • Vitamin D Sigma Aldrich, Cat. No. D1530
  • Corn Oil Sigma Aldrich, Cat. No. C8267
  • Low Calcium Special Feed (0.2% Calcium, 1% Phosphate, 2000 U Vitamin D3/kg, Nantong Trophy Feed Technology Limited) Company, 15kg
  • calcium content determination kit Najing Institute of Bioengineering, item number C004-2
  • human plasminogen (10mg/ml, purified from healthy plasma donors).
  • Aloka Micro CT is designed for the observation of mouse and rat morphology. With the latest third-generation X-ray measurement, high-quality tomographic images can be obtained in a short time. Can be used for bone measurement (bone density, bone mineral content, bone volume, bone microstructure, etc.), body fat percentage measurement, visceral, subcutaneous fat identification and measurement, synchronous photography. Bone measurements were performed on mouse femur, cranial or lumbar vertebrae. After the mice were sacrificed, the femur, cranium and lumbar vertebrae were fixed in 4% paraformaldehyde, and bone was measured using Micro CT (Aloka, manufactured by HITACHI, Japan).
  • Example 1 plasminogen deficiency is closely related to osteoporosis
  • mice Five wild-type and plasminogen-deficient (Plg -/- ) mice were 15 weeks old. The knee joint was fixed in 4% paraformaldehyde for 24 hours, then decalcified in 10% EDTA for three weeks, and washed with a gradient sucrose solution. The above operation was carried out at 4 °C. Then paraffin-embedded, 8 ⁇ m sections were stained with Safranin O. Sections were observed under a 200x optical microscope.
  • FIG. 1B The results showed that Plg ⁇ / ⁇ mice ( FIG. 1B ) exhibited extensive osteopenia and increased bone marrow cells compared to wild-type mice ( FIG. 1A ).
  • mice Five 15-week old wild-type (wt) and plasminogen-deficient (ko) mice were used. The mice in both groups were removed from the eyeballs and blood calcium levels were measured. Under normal conditions, the body's calcium balance is very well regulated. However, in the case of osteoporosis, calcium loss is a key sign of osteoporosis.
  • Plg -/- mice We investigated the levels of calcium in wild-type and Plg -/- mice and found that Plg -/- (Ko) mice had significantly higher blood calcium levels at 15 weeks of age than wild-type mice, with statistically significant differences (* indicates P ⁇ 0.05) (Fig. 2).
  • mice Eighteen 20-week-old Plg -/- mice were randomly divided into two groups, and the vehicle PBS control group and the plasminogen group were each given 4 rats. On the first day of the experiment, the group was weighed and started to give plasminogen or vehicle PBS. The plasminogen group was administered with plasminogen at a dose of 1 mg/0.1 mL/day/day via tail vein, and the vehicle was administered to the tail vein of the PBS control group. The same volume of PBS was administered by injection. The mice were sacrificed on the 31st day for 30 days, and the knee joint was fixed in the fixing solution at 4 ° C for 24 hours.
  • Fixative formulation 2% paraformaldehyde, 0.075 mol/L lysine, 0.01 mol/L sodium periodate. After the fixation, the 4 ° C PBS washing solution was washed for 12 hours each time, and then decalcified in a decalcifying solution at 4 ° C for 2 weeks, and the decalcifying solution was changed every 5 days. After the decalcification was completed, the gradient was washed in a 4° C. PBS washing solution for 12 hours, and the knee joint was dehydrated by an alcohol gradient and transparently dipped with xylene to be paraffin-embedded.
  • the thickness of the slice was 5um, the slice was dewaxed and rehydrated, and stained with hematoxylin and eosin (H&E staining), 1% hydrochloric acid alcohol was differentiated, the ammonia water returned to blue, and the alcohol gradient was dehydrated.
  • the xylene was transparent, and the neutral gum was sealed and sliced at 200 times. Observed under an optical microscope.
  • mice Eighteen 20-week-old Plg -/- mice were randomly divided into two groups, and the vehicle PBS control group and the plasminogen group were each given 4 rats. On the first day of the experiment, the group was weighed and started to give plasminogen or vehicle PBS. The plasminogen group was administered with plasminogen at a dose of 1 mg/0.1 mL/day/day via tail vein, and the vehicle PBS control group was given the same. Volume of PBS. After 30 days of continuous administration, the mice were sacrificed at the 31st place, and the femur and the fixing solution were fixed at 4 ° C for 24 hours.
  • Fixative formulation 2% paraformaldehyde, 0.075 mol/L lysine, 0.01 mol/L sodium periodate. After the fixation, the 4 ° C PBS washing solution was washed for 12 hours each time, and then decalcified in a decalcifying solution at 4 ° C for 2 weeks, and the decalcifying solution was changed every 5 days. After the decalcification was completed, the gradient was washed in a 4° C. PBS washing solution for 12 hours, and the knee joint was dehydrated by alcohol gradient and transparent with xylene, and then paraffin-embedded. Section 5 um, dewaxed and rehydrated, and magnesium chloride buffer was incubated overnight at 4 °C.
  • the alkaline phosphatase substrate solution was incubated for 1 hour at room temperature and counterstained for 2 minutes with hematoxylin. Rinse with running water for 5 minutes, bake at 60 ° C for 30 minutes, seal with neutral gum, and observe the sections under a 200x optical microscope.
  • Alkaline phosphatase is a marker of early differentiation of osteoblasts [33] .
  • the results showed that only a small amount of alkaline phosphatase was stained on the surface of the articular cartilage in the vehicle PBS control group (Fig. 4A), but the surface of the articular cartilage in the plasminogen group (Fig. 4B) was more deep. Red alkaline phosphatase staining.
  • the alkaline phosphatase activity on the surface of articular cartilage in the plasminogen group was significantly higher than that in the control group, that is, plasminogen significantly increased the osteoblast activity of the articular cartilage of the knee joint.
  • mice Eighteen 20-week-old Plg -/- mice were randomly divided into two groups, and the vehicle PBS control group and the plasminogen group were each given 4 rats. On the first day of the experiment, the group was weighed and started to give plasminogen or vehicle PBS. The plasminogen group was administered with plasminogen at a dose of 1 mg/0.1 mL/day/day via tail vein, and the vehicle PBS control group was given the same. Volume of PBS. After 30 days of continuous administration, the mice were sacrificed at the 31st place, and the femur and the fixing solution were fixed at 4 ° C for 24 hours.
  • Fixative formulation 2% paraformaldehyde, 0.075 mol/L lysine, 0.01 mol/L sodium periodate. After the fixation, the 4 ° C PBS washing solution was washed for 12 hours each time, and then decalcified in a decalcifying solution at 4 ° C for 2 weeks, and the decalcifying solution was changed every 5 days. After the decalcification was completed, the gradient washing was carried out for 4 hours at 4 ° C in PBS, and the femur was dehydrated by alcohol gradient and transparent with xylene, followed by paraffin embedding. Section 5 um, dewaxed and rehydrated, and magnesium chloride buffer was incubated overnight at 4 °C.
  • the alkaline phosphatase substrate solution was incubated for 1 hour at room temperature and counterstained for 2 minutes with hematoxylin. Rinse with running water for 5 minutes, bake at 60 ° C for 30 minutes, seal with neutral gum, and observe the photograph under 200 times under the microscope.
  • the vehicle PBS control group (Fig. 5A) showed alkaline phosphatase staining (arrow mark) at the growth plate osteoblast activity, which was light red; the plasminogen group (Fig. 5B) was present at the growth plate. More alkaline phosphatase coloring, dark red. After the administration of plasminogen, it can promote the increase of osteoblast activity of the knee growth plate. After the administration of plasminogen, it can promote the increase of osteoblast activity of the knee growth plate.
  • mice Twenty-five male C57 mice aged 5-6 weeks were randomly divided into 3 groups, 5 in the blank control group, 10 in the plasminogen group and 10 in the vehicle control group.
  • the blank control mice were intraperitoneally injected with 50 ⁇ l of corn oil per day; the plasminogen group and the vehicle-treated PBS control mice were intraperitoneally injected with vitamin D (Sigma Aldrich) at 0.5 ⁇ g/kg/day to induce senescence [34, 35] .
  • the mice began to be administered.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • mice in the group were not administered, and were continuously administered for 28 days. During the administration period, the mice in the blank control group were fed with low-calcium feed, and the plasminogen group and the vehicle-treated PBS control mice were fed with low-calcium feed. The first model was administered on the first day. On the 29th day, the eyeball was removed and blood was taken. The supernatant was centrifuged to measure the activity of serum alkaline phosphatase (ALP).
  • ALP serum alkaline phosphatase
  • Serum ALP is an isoenzyme glycoprotein. Serum ALP is mainly derived from liver and bone. The ALP derived from bone accounts for 40% to 75%. ALP activity assays are primarily used to diagnose hepatobiliary and skeletal disorders. Clinically, in addition to factors such as liver disease and pregnancy, serum ALP can also reflect osteogenesis. When bone metabolism is strong, osteoblasts are active, ALP secretion is increased, and it is present around the osteoblasts and on the surface. It is easily released into the blood, which increases the serum ALP activity. Therefore, serum ALP is a sign of changes in bone remodeling activity. One [36] .
  • mice Fifteen male C57 mice aged 5-6 weeks were randomly divided into 3 groups, the blank control group, the plasminogen group and the vehicle control PBS control group, 5 in each group.
  • the blank control mice were intraperitoneally injected with 50 ⁇ l of corn oil per day; the plasminogen group and the vehicle-treated PBS control mice were intraperitoneally injected with vitamin D (Sigma Aldrich) at 1 ⁇ g/kg/day to induce senescence [34,35] .
  • the mice began to be administered.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • mice in the group were not administered, and were continuously administered for 28 days. All mice were fed a low calcium diet (Nantong Tenofi) during dosing.
  • the first model administration was started on the first day, and on the 29th day, the knee joints were sacrificed and fixed in the fixing solution for 24 hours.
  • Fixative formulation 2% paraformaldehyde, 0.075 mol/L lysine, 0.01 mol/L sodium periodate.
  • the 4 ° C PBS washing solution was washed for 12 hours each time, and then decalcified in a decalcifying solution at 4 ° C for 2 weeks, and the decalcifying solution was changed every 5 days. After the decalcification was completed, the gradient was washed in a 4° C.
  • PBS washing solution for 12 hours, and the knee joint was dehydrated by alcohol gradient and transparent with xylene, and then paraffin-embedded. Section 5 um, dewaxed and rehydrated, and magnesium chloride buffer was incubated overnight at 4 °C.
  • the alkaline phosphatase substrate solution was incubated for 1 hour at room temperature and counterstained for 2 minutes with hematoxylin. Rinse with running water for 5 minutes, bake at 60 ° C for 30 minutes, seal with neutral gum, and observe the sections under a 200x optical microscope.
  • mice Five Plg +/+ mice and five Plg ⁇ / ⁇ mice of 12-13, 20-21, and 29-30 weeks old were used, and the weight of each group of mice was basically the same. Mice were fed the same food and water during the experiment. The skull was fixed in 4% paraformaldehyde for 24 hours, and a micro CT scan was performed to determine the bone density.
  • Osteoporosis is a systemic bone disease characterized by decreased bone mass and degeneration of bone microstructure, resulting in increased bone fragility and easy fracture.
  • the WHO recommends the use of bone mineral density (BMD) measurements to diagnose osteoporosis [37,38] .
  • BMD bone mineral density
  • mice of 20-21 weeks old, Plg +/+ mice and Plg -/- mice were used, and the mice were basically the same weight. Mice were fed the same food and water during the experiment. The skull was fixed in 4% paraformaldehyde for 24 hours, and a micro CT scan was performed to determine the bone mineral content.
  • mice Five Plg +/+ mice and five Plg ⁇ / ⁇ mice of 12-13, 20-21, and 29-30 weeks old were used, and the weight of each group of mice was basically the same. Mice were fed the same food and water during the experiment. The femur was fixed in 4% paraformaldehyde for 24 hours, and a micro CT scan was performed to determine the bone density.
  • the femur density of Plg +/+ mice increased gradually with the increase of age during the period of 12-30 weeks, while the bone density (10A) and cancellous bone density of femur cortex in Plg -/- mice (Fig. 10B)
  • the trabecular bone density (Fig. 10C) and the total bone density (Fig. 10D) were gradually reduced.
  • the femur density of Plg +/+ mice was higher than that of Plg -/- mice, and the bone density of the two strains was significantly different at 20 weeks of age, and the difference between the two became more and more different with the increase of the age.
  • plasminogen is involved in the regulation of femoral mineral metabolism and plays an important role in a certain period of time.
  • mice Five Plg +/+ mice and five Plg ⁇ / ⁇ mice of 12-13, 20-21, and 29-30 weeks old were used, and the weight of each group of mice was basically the same. Mice were fed the same food and water during the experiment. The femur was fixed in 4% paraformaldehyde for 24 hours, and a micro CT scan was performed to determine the bone mineral content.
  • Example 12 plasminogen deficient mouse lumbar vertebrae bone density reduction
  • mice Five Plg +/+ mice and five Plg ⁇ / ⁇ mice of 12-13, 20-21, and 29-30 weeks old were used, and the weight of each group of mice was basically the same. Mice were fed the same food and water during the experiment. The lumbar vertebrae were fixed in 4% paraformaldehyde for 24 hours, and a micro CT scan was performed to determine the bone density.
  • Example 13 Loss of mineral content in lumbar vertebrae of plasminogen-deficient mice
  • mice Five Plg +/+ mice and five Plg ⁇ / ⁇ mice of 12-13, 20-21, and 29-30 weeks old were used, and the weight of each group of mice was basically the same. Mice were fed the same food and water during the experiment. The lumbar vertebrae were fixed in 4% paraformaldehyde for 24 hours, and a micro CT scan was performed to determine the bone mineral content.
  • mice Five Plg +/+ mice and five Plg ⁇ / ⁇ mice of 12-13, 20-21, and 29-30 weeks old were used, and the weight of each group of mice was basically the same. Mice were fed the same food and water during the experiment. All mice were removed from the eyeballs and the supernatant was centrifuged. Serum alkaline phosphatase activity was measured using an alkaline phosphatase assay kit.
  • mice Thirteen of the 6-week-old ApoE male mice were fed a high-fat, high-cholesterol diet for 16 weeks to induce atherosclerosis [39,40] .
  • Three days before the administration 50 ⁇ L of blood was taken from each mouse, and the total cholesterol concentration was measured, and the mice were randomly divided into two groups, 7 cells in the vehicle PBS control group and 6 cells in the plasminogen group.
  • the first dose was started on the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice continued to feed high fat diets during the period. On the 30th day, the mice were fasted for 16 hours.
  • the blood calcium test was carried out using a calcium detection kit (Nanjing Institute of Bioengineering, item number C004-2) and tested according to the instructions.
  • mice Thirteen of the 6-week-old ApoE male mice were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce atherosclerosis [39,40] .
  • a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce atherosclerosis [39,40] .
  • 50 ⁇ L of blood was taken from each mouse, and the total cholesterol concentration was measured, and the mice were randomly divided into two groups, 7 cells in the vehicle PBS control group and 6 cells in the plasminogen group.
  • the first dose was started on the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice continued to feed high fat diets during the period.
  • mice were sacrificed on the 31st day of administration, and the hearts were fixed in 4% paraformaldehyde for 24-48 hours, 15%, 30% sucrose dehydrated, embedded in OCT, frozen section thickness 8 ⁇ m, alizarin red S staining 3 minute. Sections were observed under a 40x optical microscope.
  • mice 19-week-old AopE male mice were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce atherosclerosis [39,40] .
  • a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce atherosclerosis [39,40] .
  • 50 ⁇ L of blood was taken from each mouse, and the total cholesterol concentration was measured, and the mice were randomly divided into two groups, and 9 rats in the vehicle PBS control group and 10 in the plasminogen group were administered.
  • the first dose was started on the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice continued to feed high fat diets during the period.
  • mice On the 11th day of administration, 5 mice were taken from each group, and after sacrifice, the femur was fixed in 4% paraformaldehyde. The remaining mice were sacrificed 31 days after administration, and the femur was fixed in 4% paraformaldehyde. The femur was taken for micro CT scan to determine bone density.
  • Atherosclerosis has long been reported, and hyperlipidemia is an important cause of atherosclerosis.
  • apolipoprotein E (ApoE) not only affects lipid metabolism, but also is associated with bone mineral density, bone loss, and osteoporotic fractures [41, 42] .
  • vascular calcification is the transformation of vascular smooth muscle cells into osteoblast phenotype and the transformation of vascular tissue into bone tissue.
  • the formation of vascular calcification is also significantly associated with bone mineral loss [10] .
  • plasminogen can enhance bone density while reducing calcium deposition in the arterial wall. It is of great significance for the prevention and treatment of osteoporosis and cardiovascular diseases.
  • 50 ⁇ L of blood was taken from each mouse, and the total cholesterol concentration was measured, and the mice were randomly divided into two groups, three in the vehicle PBS control group and four in the plasminogen group.
  • the first dose was started on the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • mice were sacrificed at 31 days of dosing and the femurs were fixed in 4% paraformaldehyde. It was then decalcified with an acidic decalcifying solution (a decalcifying solution of 8% hydrochloric acid and 10% formic acid in ultrapure water) for 3.5 hours. Then, paraffin-embedded, 8 ⁇ m sections were subjected to H&E staining, and the sections were observed under a light microscope of 100 times (A, D) and 200 times (B, C, E, F).
  • A, D 8% hydrochloric acid and 10% formic acid in ultrapure water
  • the vehicle PBS control group (Fig. 18A-C) showed mild fibrosis of the cartilage surface (marked by thin arrows), the trabecular bone (triangular marker) was thinned, the thickness was uneven, and the cartilage tissue (star mark) was disorderly arranged.
  • the growth plate (thick arrow mark) is disordered, the chondrocytes are slightly reduced, and the tidal line is basically clear; the surface of the articular cartilage is basically normal to the plasminogen group (Fig. 18D-F), the tidal line is clear, and the trabecular bone thickness is uniform.
  • the growth plate has a clear structure and can be divided into rules. This indicates that plasminogen can improve the condition of knee joint in ApoE atherosclerotic model mice.
  • mice of 8-10 weeks old were weighed, and the mice were randomly divided into two groups according to body weight, 3 in the normal control group and 14 in the model group.
  • the mice in the model group were anesthetized with 50 mg/kg body weight by intraperitoneal injection of sodium pentobarbital.
  • the mice were detached from the hair on both sides of the back with 70% alcohol and iodine disinfection.
  • the skin, back muscles and peritoneum were cut open and gently smear with a small forceps.
  • the white shiny cellulite is pulled out of the incision, and the cellulite is separated to reveal the ovary.
  • the lower part of the ovary is first ligated with a silk thread, and then the ovaries are removed.
  • mice After the incision is sutured, the external application of anti-inflammatory powder. The same method was used to remove the other side of the ovaries. Normal control mice were only cut at the same location and no ovariectomy was performed. After 14 days of oophorectomy, the mice in the model group were randomly divided into two groups according to body weight, and given to the plasminogen group and the vehicle-controlled PBS control group, 7 in each group. The mice in the model group were induced by intraperitoneal injection of dexamethasone at a dose of 125 ⁇ g/week for 5 days/week for 12 days (43) . The normal control group was not injected. The mice were started to be injected with dexamethasone.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the cells in the normal control group were not injected with plasminogen or PBS for 16 days.
  • the start of administration was determined to be the first day, and the body weight of the mice was measured on the 17th day.
  • mice 14 of C57 female mice 8-10 weeks old were weighed. All mice were anesthetized with 50 mg/kg body weight by intraperitoneal injection of sodium pentobarbital. The mice were detached from the back of the hair with 70% alcohol and iodine disinfection. The skin, back muscles and peritoneum were cut open, and the whites were gently white with a small forceps. The ovary is visible when the cellulite is pulled out of the incision and the cellulite is separated. The lower part of the ovary is first ligated with a silk thread, and then the ovaries are removed. After the incision is sutured, the external application of anti-inflammatory powder. The same method was used to remove the other side of the ovaries.
  • mice After 14 days of oophorectomy, the mice were randomly divided into two groups according to body weight, and given to the plasminogen group and the vehicle PBS control group, 7 in each group. The two groups of mice were induced to have osteoporosis by intraperitoneal injection of dexamethasone at a dose of 5 days/week for 12 days. [43] At the same time of modeling, the mice were started to be administered. The plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. Drug for 16 days. The first dose was started on the first day, and the femur was sacrificed in the 4% paraformaldehyde fixative on the 17th day. A Micro CT scan was performed to measure the femoral indicators.
  • mice of 8-10 weeks old were weighed, and the mice were randomly divided into two groups according to body weight, 3 in the normal control group and 14 in the model group.
  • the mice in the model group were anesthetized with 50 mg/kg body weight by intraperitoneal injection of sodium pentobarbital.
  • the mice were detached from the hair on both sides of the back with 70% alcohol and iodine disinfection.
  • the skin, back muscles and peritoneum were cut open and gently smear with a small forceps.
  • the white shiny cellulite is pulled out of the incision, and the cellulite is separated to reveal the ovary.
  • the lower part of the ovary is first ligated with a silk thread, and then the ovaries are removed.
  • mice in the model group were induced with osteoporosis by intraperitoneal injection of dexamethasone at a dose of 5 days/week for 12 days (43) .
  • the normal control group was not treated with injection.
  • the mice in the model group were randomly divided into two groups according to their body weight, and given to the plasminogen group and the vehicle-controlled PBS control group, 7 rats in each group.
  • mice After the model was established (the day after the completion of dexamethasone injection), the mice started to be administered.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the PBS control group for 16 days.
  • the normal control group of mice Do not inject plasminogen or PBS.
  • the first dose was started on the first day, and the femur was sacrificed in the 4% paraformaldehyde fixative on the 17th day.
  • the femur obtained was subjected to a Micro CT scan to determine the femur density.
  • plasminogen can significantly improve osteoporosis, promote bone mineral density and bone mass increase in various parts of the femur, and the improvement of trabecular bone is particularly obvious.
  • mice 14 of C57 female mice 8-10 weeks old were weighed. All mice were anesthetized with 50 mg/kg body weight by intraperitoneal injection of sodium pentobarbital. The mice were detached from the back of the hair with 70% alcohol and iodine disinfection. The skin, back muscles and peritoneum were cut open, and the whites were gently white with a small forceps. The ovary is visible when the cellulite is pulled out of the incision and the cellulite is separated. The lower part of the ovary is first ligated with a silk thread, and then the ovaries are removed. After the incision is sutured, the external application of anti-inflammatory powder. The same method was used to remove the other side of the ovaries.
  • mice After 14 days of oophorectomy, the mice were randomly divided into two groups according to body weight, and given to the plasminogen group and the vehicle PBS control group, 7 in each group. The two groups of mice were induced to have osteoporosis by intraperitoneal injection of dexamethasone at a dose of 5 days/week for 12 days. [43] At the same time of modeling, the mice were started to be administered. The plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. Drug for 16 days.
  • the first dose was started on the first day, and the knee joint was sacrificed on the 17th day and fixed in 4% paraformaldehyde fixative. It was then decalcified with an acidic decalcifying solution (a decalcifying solution of 8% hydrochloric acid and 10% formic acid in ultrapure water) for 3.5 hours. Then, paraffin-embedded, 3 ⁇ m sections were stained with H&E (A, B) and Safrain O (C, D), and sections were observed under a 100-fold optical microscope.
  • an acidic decalcifying solution a decalcifying solution of 8% hydrochloric acid and 10% formic acid in ultrapure water
  • Example 23 Increased plasminogen activity in ovariectomy-dexamethasone osteoporosis model mouse knee joint osteoblasts
  • mice 14 of C57 female mice 8-10 weeks old were weighed. All mice were anesthetized with 50 mg/kg body weight by intraperitoneal injection of sodium pentobarbital. The mice were detached from the back of the hair with 70% alcohol and iodine disinfection. The skin, back muscles and peritoneum were cut open, and the whites were gently white with a small forceps. The ovary is visible when the cellulite is pulled out of the incision and the cellulite is separated. The lower part of the ovary is first ligated with a silk thread, and then the ovaries are removed. After the incision is sutured, the external application of anti-inflammatory powder. The same method was used to remove the other side of the ovaries.
  • mice After 14 days of oophorectomy, the mice were randomly divided into two groups according to body weight, and given to the plasminogen group and the vehicle PBS control group, 7 in each group. The two groups of mice were induced to have osteoporosis by intraperitoneal injection of dexamethasone at a dose of 5 days/week for 12 days. [43] At the same time of modeling, the mice were started to be administered. The plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. Drug for 16 days.
  • the first dose was started on the first day, and the knee joint was sacrificed on the 17th day to fix the knee joint in the fixative.
  • Fixative formulation 2% paraformaldehyde, 0.075 mol/L lysine, 0.01 mol/L sodium periodate.
  • the 4 ° C PBS washing solution was washed for 12 hours each time, and then decalcified in a decalcifying solution at 4 ° C for 2 weeks, and the decalcifying solution was changed every 5 days.
  • the gradient was washed in a 4° C. PBS washing solution for 12 hours, and the knee joint was dehydrated by alcohol gradient and transparent with xylene, and then paraffin-embedded.
  • Section 3um, dewaxed rehydration, and magnesium chloride buffer was incubated overnight at 4 °C.
  • the alkaline phosphatase substrate solution was incubated for 1 hour at room temperature and counterstained for 2 minutes with hematoxylin. Rinse with running water for 5 minutes, bake at 60 ° C for 30 minutes, seal with neutral gum, and observe the sections under a 200x optical microscope.
  • Example 24 plasminogen reduces blood calcium concentration in ovariectomized osteoporosis model mice
  • mice 11 8-10 week old Plg +/+ female mice.
  • the mice were anesthetized with 50 mg/kg body weight by intraperitoneal injection of sodium pentobarbital.
  • the mice were detached from the hair on both sides of the back with 70% alcohol and iodine.
  • the skin, back muscles and peritoneum were cut, and the white hair was gently rubbed with a small forceps.
  • the bright cellulite is pulled out of the incision, and the cellulite is separated to reveal the ovary.
  • the lower part of the ovary is first ligated with a silk thread, and then the ovaries are removed. After the incision is sutured, the external application of anti-inflammatory powder.
  • the same method removed the other side of the ovary [44,45] .
  • mice were weighed and randomly divided into two groups according to body weight, 6 rats in the plasminogen group and 5 rats in the vehicle control group, and administration was started.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group for 11 days.
  • the first dose was started on the first day.
  • the eyeballs were removed, blood was taken, and the supernatant was centrifuged to measure the blood calcium concentration.
  • the blood calcium test was carried out using a calcium detection kit (Nanjing Institute of Bioengineering, item number C004-2) and tested according to the instructions.
  • Example 25 Increased plasma phosphatase concentration in ovariectomized osteoporosis model mice by plasminogen
  • mice 11 8-10 week old Plg +/+ female mice.
  • the mice were anesthetized with 50 mg/kg body weight by intraperitoneal injection of sodium pentobarbital.
  • the mice were detached from the hair on both sides of the back with 70% alcohol and iodine.
  • the skin, back muscles and peritoneum were cut, and the white hair was gently rubbed with a small forceps.
  • the bright cellulite is pulled out of the incision, and the cellulite is separated to reveal the ovary.
  • the lower part of the ovary is first ligated with a silk thread, and then the ovaries are removed. After the incision is sutured, the external application of anti-inflammatory powder. Removal of the ovaries same method the other side [44,45].
  • mice After 65 days of ovariectomy, the mice were weighed and randomly divided into two groups according to body weight, 6 rats in the plasminogen group and 5 rats in the vehicle control group, and administration was started.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group for 11 days.
  • the first dose was started on the first day. On the 12th day, the eyeballs were removed and blood was taken. The supernatant was centrifuged to measure the blood phosphorus concentration. Blood phosphorus detection was carried out using a phosphorus detection kit (Nanjing Institute of Bioengineering, item number C006-3) and tested according to the instructions.
  • mice of 9 weeks old were fed 3% cholesterol high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia [46,47] .
  • This model was designated as a model of 3% cholesterol hyperlipidemia.
  • the modeled mice continued to be fed a 3% cholesterol high fat diet.
  • 50 ⁇ L of blood was taken from each mouse three days before the administration, and total cholesterol was measured, and was randomly divided into two groups according to the total cholesterol concentration and body weight, with 8 in each group. The first dose was recorded as the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 20 days.
  • the mice were fasted for 16 hours, and on the 21st day, the mice were sacrificed and the knee joints were fixed in the fixative.
  • Fixative formulation 2% paraformaldehyde, 0.075 mol/L lysine, 0.01 mol/L sodium periodate.
  • the 4 ° C PBS washing solution was washed for 12 hours each time, and then decalcified in a decalcifying solution at 4 ° C for 2 weeks, and the decalcifying solution was changed every 5 days.
  • the gradient was washed in a 4° C. PBS washing solution for 12 hours, and the knee joint was dehydrated by alcohol gradient and transparent with xylene, and then paraffin-embedded.
  • Section 3um, dewaxed rehydration, and magnesium chloride buffer was incubated overnight at 4 °C.
  • the alkaline phosphatase substrate solution was incubated for 1 hour at room temperature and counterstained for 2 minutes with hematoxylin. Rinse with running water for 5 minutes, bake at 60 ° C for 30 minutes, seal with neutral gum, and observe the sections under a 200x optical microscope.
  • Hyperlipidemia is a disorder in which lipid metabolism is disordered and can cause a series of complications. In recent years, several studies have found that hyperlipidemia is a common cause of osteoporosis and atherosclerosis [48,49] .
  • mice 14 of C57 female mice 8-10 weeks old were weighed.
  • the mice were anesthetized with 50 mg/kg body weight by intraperitoneal injection of sodium pentobarbital.
  • the mice were detached from the hair on both sides of the back with 70% alcohol and iodine.
  • the skin, back muscles and peritoneum were cut, and the white hair was gently rubbed with a small forceps.
  • the bright cellulite is pulled out of the incision, and the cellulite is separated to reveal the ovary.
  • the lower part of the ovary is first ligated with a silk thread, and then the ovaries are removed. After the incision is sutured, the external application of anti-inflammatory powder. The same method was used to remove the other side of the ovaries.
  • mice in the model group were induced to have osteoporosis by intraperitoneal injection of dexamethasone at a dose of 5 days/week for 12 days [43] .
  • the mice were randomly divided into two groups according to their body weight, and given to the plasminogen group and the vehicle PBS control group, 7 rats in each group.
  • the mice started to be administered.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group for 16 days.
  • the first dose was started on the first day, and the knee joint was sacrificed on the 17th day and fixed in 4% paraformaldehyde fixative. It was then decalcified with an acidic decalcifying solution (a decalcifying solution of 8% hydrochloric acid and 10% formic acid in ultrapure water) for 3.5 hours. Then, paraffin-embedded, 3 ⁇ m sections were stained with H&E (A, B) and Safrain O (C, D), and sections were observed under a 100-fold optical microscope.
  • an acidic decalcifying solution a decalcifying solution of 8% hydrochloric acid and 10% formic acid in ultrapure water
  • trabecular bone (arrow mark) of the vehicle PBS control group (Fig. 27A, C) was significantly thinned and fractured, and a large area of the trabecular bone marrow cavity was absent, and the connection of the trabecular bone was interrupted. Partial fibrosis, the osteogenesis of the osteogenesis area under the growth plate was significantly reduced (triangular marker); the trabecular bone fraction of the plasminogen group (Fig. 27B, D) was thinner, and the trabecular bone was continuous compared with the PBS control group. The sex is better, there is no serious fracture, there is no large area of the trabecular trabecular region, the cartilage tissue hierarchy is more regular, and the tide line is clear. It is indicated that the administration of plasminogen can significantly improve the tissue structure of knee joints in osteoporosis model mice.
  • Stoppelli M. P., Corti, A., Soffientini, A., Cassani, G., Blasi, F., and Assoian, RK (1985) .

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Abstract

本发明提供了纤溶酶原用于预防和/或治疗骨质疏松及其相关病症的用途。本发明还提供了预防和/或治疗骨质疏松的药物及制品。

Description

一种预防和治疗骨质疏松的药物及其用途 技术领域
本发明涉及纤溶酶原用于预防或治疗骨质疏松及相关疾病的用途。
背景技术
骨质疏松症(osteoporosis，OP)是一种以骨量减少和骨组织微结构破坏为特征,并能导致骨脆性增加和易于骨折的全身性疾病。2001年美国国立卫生研究院(NIH)提出骨质疏松症是以骨强度下降、骨折风险性增加为特征的骨骼系统疾病，骨强度反映骨骼的两个主要方面，即骨密度和骨质量。骨质疏松症导致骨量减少和骨微结构的退化，使得患者骨骼的脆性增加，严重地降低患者的运动功能和生活质量。
哺乳动物骨骼发育是一个高度有序，受多重因素共同调控的过程。哺乳动物的骨骼发育主要通过膜内成骨和软骨内成骨两种方式完成，其中四肢骨、椎骨等长骨主要通过软骨内成骨形成，颅骨、锁骨内侧部分等扁骨则是通过膜内成骨[1]。骨组织形成后并非一成不变，而是处于骨形成与吸收的稳态动态平衡中。在此动态平衡的过程中，激素、多种信号通路、骨组织细胞的协同调控及矿物盐的稳态发挥着重要作用[2]。
骨质疏松大致可分为原发性和继发性两大类，而绝经后骨质疏松和老年性骨质疏松均属于原发性骨质疏松，且十分常见。继发性骨质疏松症是一种常见的全身性骨病，除了已知的疾病和诱发骨质疏松的药物之外，一些新兴的药物和治疗手段都已经成为继发性骨质疏松症的重要病因。
流行病学调查显示，1型糖尿病患者骨量减少和骨质疏松发病率为48％～72％[3]，对于2型糖尿病患者，骨密度增高、降低或没有改变的结果国内外文献均可见报道[4-6]。近年来，研究发现2型糖尿病患者代谢性骨病的发病率和骨质疏松性骨折的危险性明显高于普通人群，其骨质疏松发生率可达20％～60％[5]。糖尿病骨质疏松易导致病理性骨折，致残致死率高，可加重糖尿病患者的治疗和康复困难。
在经过了一个多世纪的观察中，人们发现骨质疏松患者常常因合并心肌梗死、中风、猝死而使死亡率明显上升；而有动脉粥样硬化的患者也常常合并骨量的丢失，导致骨质疏松性骨折的发生[7-9]。过去常常认为动脉粥样硬化与骨质疏松是随年龄增加而出现的退行性改变，但随着对二者长期的临床观察及分子机制的深入研究，人们发现:①二者存在共同的危险因素如衰老、糖尿血管细胞中的血管钙化细胞(calcifying vascularcells，CVC)其分子特征与骨生物学特征之间越来越一致和平行，在敲除骨代谢相关基因的动物出现了血管特征性表现，提示它们之间有共同的信号途径、转录因子和细胞外基质的相互作用；③活性氧(reactive oxygen species，ROS)以及氧化型脂质对血管及骨骼共同影响；④内分泌异常如雌激素的减少、甲状旁腺激素(parathyroid hormone，PTH)和维生素D、降钙素代谢异常；⑤这两个疾病的治疗策略方面也存在密切的联系。随着人们对这两个看似矛盾、但常在同一机体发生的疾病的机制进一步了解，对As/OP综合征防治也不断地深入。
近年来的多项研究显示心血管系统疾病和骨质疏松症存在相关性，它们共同发生在老年期，经常在同一老年个体可见，且发病率都随着年龄的增长而增加。尽管老年是心血管疾病和骨质疏松共同的危险因素，但大多数研究发现，忽略年龄这一因素后，这两种疾病之间仍存在显著的联系。一方面，心血管系统疾病与骨量丢失及骨折风险增加有关，同样，有证据提示骨密度降低可导致心血管系统疾病的发病率和死亡率增加。进一步研究发现心血管疾病与骨质疏松在发病机制层面有紧密而直接的联系。动脉粥样硬化是心脑血管疾病的主要病理基础，而动脉钙化则是其主要表现之一。动脉钙化被认为是心血管疾病的一个重要标志物和临床监测指标。研究表明，血管钙化的本质是血管平滑肌细胞向成骨细胞表型的转化以及血管组织向骨组织的转化。而血管钙化的形成也和骨矿物质丢失具有显著的相关性。第三军医大学周锐博士[10]对一组60岁以上老年人群进行了观察性研究，探讨了老年患者动脉钙化和骨质疏松及骨折之间相关性。他的研究包括：1.从2012年1月1日到12月31日，筛选60岁以上符合条件的来院就诊患者为研究对象。2.主动脉钙化程度的半定量测量:应用腰椎x线侧位片对第1-4腰椎对应的腹主动脉钙化沉积物进行评分。根据钙化斑块的长度和累及的 节段数，每位患者的主动脉钙化评分(acs)为0-24分不等，无主动脉钙化是0分，最严重的主动脉钙化是24分。并依据acs将患者分为进行了分组。3.骨密度检测使用双能x线吸收仪测量(dxa)。骨质疏松症定义为基于dxa测定的骨密度值低于同性别、同种族正常成年人骨峰值2.5个标准差或以上。4.采用多因素回归风险模型评估主动脉钙化和骨质疏松症发生风险的关系。同时，该研究还包括：1.筛选60岁以上符合条件的绝经后妇女为研究对象。2.主动脉钙化程度的半定量测量。3.椎骨骨折的诊断:通过x线片观察椎体形态(胸4-腰5节段)确定椎骨骨折的发生(椎体高度下降20％以上)。4.采用多因素回归分析模型评估主动脉钙化和椎骨骨折之间的关系。同时，该研究还包括1.筛选60岁以上符合条件的来院就诊患者为研究对象。2.骨密度检测及骨质疏松症的诊断。3.颈动脉和冠状动脉粥样硬化钙化斑块检测:使用64排螺旋ct行颈动脉和冠状动脉cta。三维图像分析工作站评估所有cta图像。并对动脉斑块的成分和发生范围进行评价。4.采用多因素回归风险模型分析骨质疏松和骨量丢失与颈动脉和冠状动脉钙化斑块发生风险之间关系。研究结果发现，在调整年龄等其它混杂因素后，严重的骨量丢失与颈动脉斑块、冠状动脉斑块及共存的钙化斑块的发生均显著相关。结论是：在老年人群中，严重的主动脉钙化与骨质疏松症的发生相关。骨质疏松症的发生风险随着动脉粥样硬化的增加而升高。骨密度降低和血25(OH)D水平下降也与骨质疏松症发生的相关。在老年绝经后妇女中，严重的主动脉钙化与椎骨骨折的发生相关。骨密度降低和血25(OH)D水平下降与椎骨骨折的发生相关。在老年人群中,骨质疏松症、低骨量及血25(OH)D水平下降与动脉钙化斑块的发生相关；严重的骨量丢失与颈动脉斑块、冠状动脉斑块及共存的钙化斑块的发生风险均相关。该研究对一组老年人群的观察性研究揭示了老年病的一些共同危险因素及内在的相互关系，对骨质疏松症及心血管疾病的防治具有重要意义。
骨质疏松症是衰老相关病症中的代表性症状之一，在中老年人群尤其普遍。骨质疏松是生物衰老在骨骼方面的一种特殊表现，已经表明维生素D水平过低或者过高都与骨质疏松有关[11]。随着老龄化社会的到来，骨质疏松的发病率呈逐年上升趋势，由此所带来的社会经济负担也在大大增加。
对于骨质疏松的治疗而言，关键是恢复并维持正常的骨组织含量和降低骨折的发生率。虽然治疗的方法较多，但目前仍以药物治疗为主。常用的药物有骨吸收抑制剂、骨形成促进剂和骨矿化物。骨吸收抑制剂是主要针对破骨细胞的药物，通过抑制破骨细胞的活动而减少骨的重吸收；骨形成促进剂则是主要针对成骨细胞的药物，能够增强成骨细胞的活性,以促进新骨的合成；骨矿化物是治疗骨质疏松的基础用药,包括钙剂和维生素D，可以起到补充骨基质成分的作用。但当前治疗骨质疏松的药物绝大多数为骨吸收抑制剂(如雌激素、双膦酸盐、降钙素等)，而骨形成促进剂(如甲状旁腺激素等)的种类却非常少。从药物的治疗效果来看，如今只是停留于改善症状，延缓病情发展的水平，却并没有达到逆转病情甚至治愈的效果，因此需要寻找新的治疗药物和治疗方法。
发明简述
本发明涉及：
1. 一种预防和治疗骨质疏松及其相关病症的方法，包括给药受试者治疗有效量的纤溶酶原。
2. 项1的方法，其中所述骨质疏松包括原发性骨质疏松和继发性骨质疏松。
3. 项1的方法，其中所述原发性骨质疏松包括绝经后骨质疏松和老年性骨质疏松。
4. 项1或2的方法，其中所述继发性骨质疏松包括继发于内分泌疾病、风湿性疾病、胃肠道疾病，以及药物治疗引起的骨质疏松。
5. 项4的方法，其中所述继发性骨质疏松包括糖皮质激素、原发性甲状旁腺功能亢进、甲状腺功能亢进、原发性胆汁性肝硬化、性腺功能减低、糖尿病、高血压、动脉粥样硬化、慢性肾脏疾病、类风湿关节炎、系统性红斑狼疮、强直性脊柱炎、骨关节炎、性腺激素治疗、抗癫痫药治疗、化疗药物治疗引起的骨质疏松。
6. 一种预防和治疗疾病并发的骨质疏松的方法，包括给药受试者有效量的纤溶酶原，其中所述疾病并发的骨质疏松包括糖皮质激素、原发性甲状旁腺功能亢进、甲状腺功能亢进、原发性胆汁性肝硬化、性腺功能 减低、糖尿病、高血压、动脉粥样硬化、慢性肾脏疾病、类风湿关节炎、系统性红斑狼疮、强直性脊柱炎、骨关节炎、性腺激素治疗、抗癫痫药治疗、化疗药物治疗并发的骨质疏松。。
7. 一种预防骨质疏松骨折的方法，包括给药易患骨质疏松的受试者、处于患骨质疏松高风险的受试者或诊断患有骨质疏松的受试者有效量的纤溶酶原预防骨折的发生。
8. 项7的方法，其中所述受试者包括患有糖皮质激素、原发性甲状旁腺功能亢进、甲状腺功能亢进、原发性胆汁性肝硬化、性腺功能减低、糖尿病、高血压、动脉粥样硬化、慢性肾脏疾病、类风湿关节炎、系统性红斑狼疮、强直性脊柱炎或骨关节炎的受试者。
9. 项7的方法，其中所述受试者包括正接受性腺激素治疗、抗癫痫药治疗或化疗药物治疗的受试者。
10. 一种增强成骨细胞活性的方法，包括给药受试者有效量的纤溶酶原。
11. 一种调控骨矿物质代谢的方法，包括给药受试者有效量的纤溶酶原。
12. 项11的方法，所述调控包括降低血钙水平、升高血磷水平，促进钙在骨基质中的沉积和/或降低钙在血管壁、内脏的沉积。
13. 项1-12任一项的方法，其中所述纤溶酶原与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。
14. 项1-12任一项的方法，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。
15. 项1-12任一项的方法，所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。
16. 项1-12任一项的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。
17. 项1-12任一项的方法，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。
18. 项1-12任一项的方法，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。
19. 项13-18任一项的方法，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。
20. 项1-19任一项的方法，其中所述纤溶酶原是人天然纤溶酶原。
21. 项1-20任一项的方法，其中所述受试者是人。
22. 项1-21任一项的方法，其中所述受试者缺乏或缺失纤溶酶原。
23. 项22的方法，其中所述缺乏或缺失是先天的、继发的和/或局部的。
24. 一种用于项1-23任一项的方法的纤溶酶原。
25. 一种药物组合物，其包含药学上可接受的载剂和用于项1-23中任一项所述方法的纤溶酶原。
26. 一种预防性或治疗性试剂盒，其包含：(i)用于项1-23中任一项所述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)。
27. 根据项26所述的试剂盒，其中所述构件为注射器或小瓶。
28. 项26或27的试剂盒，其还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施项1-23中任一项所述方法。
29. 一种制品，其包含：
含有标签的容器；和
包含(i)用于项1-23中任一项所述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施项1-23中任一项所述方法。
30. 项26-28中任一项的试剂盒或项29的制品，还包含另外的一个或多个构件或容器，该构件或容器中含有其它药物。
31. 项30的试剂盒或制品，其中所述其它药物包括治疗骨质疏松的其它药物或治疗与骨质疏松并发的其它疾病的药物。
32. 包含纤溶酶原的用于治疗骨质疏松的药剂。
33. 包含纤溶酶原的用于治疗骨质疏松的药物组合物，试剂盒、制品。
34. 纤溶酶原用于治疗骨质疏松的用途。
35. 本发明还涉及纤溶酶原在制备上述项1-23任一项的方法中使用的药物、药物组合物、制品、试剂盒中的用途。
在本发明的上述任一实施方案中，所述纤溶酶原可与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。在一些实施方案中，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。
在一些实施方案中，所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。在一些实施方案中，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。在一些实施方案中，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。在一些实施方案中，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。在一些实施方案中，所述纤溶酶原是人天然纤溶酶原。
在一些实施方案中，所述受试者是人。在一些实施方案中，所述受试者缺乏或缺失纤溶酶原。在一些实施方案中，所述缺乏或缺失是先天的、继发的和/或局部的。
在一些实施方案中，所述药物组合物包含药学上可接受的载剂和用于前述方法的纤溶酶原。在一些实施方案中，所述试剂盒可以是预防性或治疗性试剂盒，其包含：(i)用于前述方法的纤溶酶原和(ii)用于递送所述纤溶 酶原至所述受试者的构件(means)。在一些实施方案中，所述构件为注射器或小瓶。在一些实施方案中，所述试剂盒还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施前述任一方法。
在一些实施方案中，所述制品包含：含有标签的容器；和包含(i)用于前述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施前述任一方法。
在一些实施方案中，所述试剂盒或制品还包含另外的一个或多个构件或容器，该构件或容器中含有其他药物。在一些实施方案中，所述其他药物选自下组：降血脂药物、抗血小板药物、降血压药物、扩张血管药物、降血糖药物、抗凝血药物、溶血栓药物，保肝药物，抗心律失常药物，强心药物，利尿药物，抗感染药物、抗病毒药物、免疫调节药物、炎症调节类药物和抗肿瘤药物。
在前述方法的一些实施方案中，所述纤溶酶原通过全身或局部给药，优选通过以下途径施用：静脉内、肌内、皮下给予纤溶酶原来进行治疗。在前述方法的一些实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在前述方法的一些实施方案中，所述纤溶酶原以每天0.0001-2000mg/kg、0.001-800mg/kg、0.01-600mg/kg、0.1-400mg/kg、1-200mg/kg、1-100mg/kg、10-100mg/kg(以每公斤体重计算)或0.0001-2000mg/cm2、0.001-800mg/cm2、0.01-600mg/cm2、0.1-400mg/cm2、1-200mg/cm2、1-100mg/cm2、10-100mg/cm2(以每平方厘米体表面积计算)的剂量施用，优选至少重复一次，优选至少每天施用。
本发明明确涵盖了属于本发明实施方案之间的技术特征的所有组合，并且这些组合后的技术方案在本申请中已经明确公开，就像上述技术方案已经单独且明确公开一样。另外，本发明还明确涵盖各个实施方案及其要素的所有亚组合，并且在本文中公开，就像每一个此类亚组合单独且明确在本文中公开一样。
发明详述
定义
“骨质疏松症”是一种以骨量低下，骨微结构损坏，导致骨脆性增加，易发生骨折为特征的全身性退化性骨病。一般分为原发性、继发性和特发性骨质疏松症3类。
“原发性骨质疏松症”又分为绝经后骨质疏松症(I型)和老年骨质疏松症(Ⅱ型)，其中绝经后骨质疏松症一般发生在妇女绝经后5～10年内；老年骨质疏松症一般指老年人60岁及以上发生的骨质疏松症。原发性骨质疏松症主要强调骨量、骨丢失与骨结构的重要作用，以骨量减少、脆性增加、结构退化、易发生骨折为其临床特征。
“继发性骨质疏松症”是指由于某些疾病、药物或其他原因造成骨量降低，骨骼的微结构发生改变，易发生脆性骨折的疾病。常见的导致骨质疏松的疾病或药物包括：
1.内分泌疾病：
库欣综合征、性腺功能减低、甲状腺功能亢进症、原发性甲状旁腺功能亢进症、糖尿病
2.风湿疾病：
类风湿关节炎、系统性红斑狼疮、强直性脊柱炎
3.血液系统疾病：
多发性骨髓瘤、白血病、淋巴瘤、地中海贫血、血友病
4.药物治疗：
糖皮质激素过量、甲状腺激素过量替代、抗癫痫药物、锂或铝中毒、细胞毒或免疫抑制剂(环孢A、他克莫司)、肝素、引起性腺功能低下的药物(芳香化酶抑制剂、促性腺激素释放激素类似物等)
5.胃肠道疾病：
慢性肝病(尤其是原发性胆汁性肝硬化)、炎性肠病(尤其是克罗恩病)、胃大部分切除术、腹泻病
6.肾脏疾病：
肾功能不全或衰竭
7.遗传性疾病
成骨不全、马凡综合征、血色病、高胱氨酸尿、卟啉病
8.其他原因：
任何原因维生素D不足、酗酒、神经性厌食、营养不良、长期卧床、妊娠及哺乳、慢性阻塞性肺疾病、脑血管意外、器官移植、淀粉样变、多发性硬化、获得性免疫缺陷综合征
这些继发性因素通过影响成骨细胞和破骨细胞功能，使骨吸收增加和/或骨形成减低而导致骨质疏松。
本发明所述的“继发性骨质疏松”的术语涵盖了上述各种原因导致的骨质疏松。
“特发性骨质疏松”主要发生在青少年中，一般指男性发病年龄小于50岁、女性发病年龄小于40岁的骨质疏松，无潜在疾病，其原因不明。
与某种疾病或状况“并发的骨质疏松”，指的是与所述疾病或状况相伴发生的骨质疏松。所述疾病或状况与骨质疏松之间可以有某种内在的病因或发病机制方面的联系。例如糖尿病并发的骨质疏松，动脉粥样硬化并发的骨质疏松，慢性肾病并发的骨质疏松、强直性脊柱炎并发的骨质疏松、骨关节炎并发的骨质疏松等。
纤溶酶是纤溶酶原激活系统(PA系统)的关键组分。它是一种广谱的蛋白酶，能够水解细胞外基质(ECM)的几个组分，包括纤维蛋白、明胶、纤连蛋白、层粘连蛋白和蛋白聚糖[12]。此外，纤溶酶能将一些金属蛋白酶前体(pro-MMPs)激活形成具有活性的金属蛋白酶(MMPs)。因此纤溶酶被认为是胞外蛋白水解作用的一个重要的上游调节物[13-14]。纤溶酶是由纤溶酶原通过两种生理性的PAs：组织型纤溶酶原激活剂(tPA)或尿激酶型纤溶酶原激活剂(uPA)蛋白水解形成的。由于纤溶酶原在血浆和其他体液中相对水平较高，传统上认为PA系统的调节主要通过PAs的合成和活性水平实现。PA系统组分的合成受不同因素严格调节，如激素、生长因子和细胞因子。此外，还存在纤溶酶和PAs的特定生理抑制剂。纤溶酶的主要抑制剂是α2-抗纤溶酶(α2-antiplasmin)。PAs的活性同时被uPA和tPA的纤溶酶原激活剂抑制剂-1(PAI-1)抑制以及主要抑制uPA的溶酶原激活剂抑制剂-2(PAI-2)调节。某些细胞表面具有直接水解活性的uPA特异性细胞表面受体(uPAR)[15-16]。
纤溶酶原是一个单链糖蛋白，由791个氨基酸组成，分子量约为92kDa[17-18]。纤溶酶原主要在肝脏合成，大量存在于胞外液中。血浆中纤溶酶 原含量约为2μM。因此纤溶酶原是组织和体液中蛋白质水解活性的一个巨大的潜在来源[19-20]。纤溶酶原存在两种分子形式：谷氨酸-纤溶酶原(Glu-plasminogen)和赖氨酸-纤溶酶原(Lys-plasminogen)。天然分泌和未裂解形式的纤溶酶原具有一个氨基末端(N-末端)谷氨酸，因此被称为谷氨酸-纤溶酶原。然而，在纤溶酶存在时，谷氨酸-纤溶酶原在Lys76-Lys77处水解成为赖氨酸-纤溶酶原。与谷氨酸-纤溶酶原相比，赖氨酸-纤溶酶原与纤维蛋白具有更高的亲和力，并可以更高的速率被PAs激活。这两种形式的纤溶酶原的Arg560-Val561肽键可被uPA或tPA切割，导致二硫键连接的双链蛋白酶纤溶酶的形成[21]。纤溶酶原的氨基末端部分包含五个同源三环，即所谓的kringles，羧基末端部分包含蛋白酶结构域。一些kringles含有介导纤溶酶原与纤维蛋白及其抑制剂α2-AP特异性相互作用的赖氨酸结合位点。最新发现一个纤溶酶原为38kDa的片段，其中包括kringles1-4，是血管生成的有效抑制剂。这个片段被命名为血管抑素，可通过几个蛋白酶水解纤溶酶原产生。
纤溶酶的主要底物是纤维蛋白，纤维蛋白的溶解是预防病理性血栓形成的关键[22]。纤溶酶还具有对ECM几个组分的底物特异性，包括层粘连蛋白、纤连蛋白、蛋白聚糖和明胶，表明纤溶酶在ECM重建中也起着重要作用[18,23-24]。间接地，纤溶酶还可以通过转化某些蛋白酶前体为活性蛋白酶来降解ECM的其他组分，包括MMP-1，MMP-2，MMP-3和MMP-9。因此，有人提出，纤溶酶可能是细胞外蛋白水解的一个重要的上游调节器[25]。此外，纤溶酶具有激活某些潜在形式的生长因子的能力[26-28]。在体外，纤溶酶还能水解补体系统的组分并释放趋化补体片段。
“纤溶酶”是存在于血液中的一种非常重要的酶，能将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体。
“纤溶酶原”是纤溶酶的酶原形式，根据swiss prot中的序列，按含有信号肽的天然人源纤溶酶原氨基酸序列(序列4)计算由810个氨基酸组成，分子量约为90kD，主要在肝脏中合成并能够在血液中循环的糖蛋白，编码该氨基酸序列的cDNA序列如序列3所示。全长的纤溶酶原包含七个结构域：位于C末端的丝氨酸蛋白酶结构域、N末端的Pan Apple(PAp)结构域以及5个Kringle结构域(Kringle1-5)。参照swiss prot中的序列，其信号肽包括 残基Met1-Gly19，PAp包括残基Glu20-Val98，Kringle1包括残基Cys103-Cys181，Kringle2包括残基Glu184-Cys262，Kringle3包括残基Cys275-Cys352，Kringle4包括残基Cys377-Cys454，Kringle5包括残基Cys481-Cys560。根据NCBI数据，丝氨酸蛋白酶域包括残基Val581-Arg804。
Glu-纤溶酶原是天然全长的纤溶酶原，由791个氨基酸组成(不含有19个氨基酸的信号肽)，编码该序列的cDNA序列如序列1所示，其氨基酸序列如序列2所示。在体内，还存在一种是从Glu-纤溶酶原的第76-77位氨基酸处水解从而形成的Lys-纤溶酶原，如序列6所示，编码该氨基酸序列的cDNA序列如序列5所示。Delta-纤溶酶原(δ-plasminogen)是全长纤溶酶原缺失了Kringle2-Kringle5结构的片段，仅含有Kringle1和丝氨酸蛋白酶域[29-30]，有文献报道了delta-纤溶酶原的氨基酸序列(序列8)[31]，编码该氨基酸序列的cDNA序列如序列7。小纤溶酶原(Mini-plasminogen)由Kringle5和丝氨酸蛋白酶域组成，有文献报道其包括残基Val443-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)[31]，其氨基酸序列如序列10所示，编码该氨基酸序列的cDNA序列如序列9所示。而微纤溶酶原(Micro-plasminogen)仅含有丝氨酸蛋白酶结构域，有文献报道其氨基酸序列包括残基Ala543-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)[32]，也有专利文献CN102154253A报道其序列包括残基Lys531-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，本专利序列参考专利文献CN102154253A，其氨基酸序列如序列12所示，编码该氨基酸序列的cDNA序列如序列11所示。
本发明的“纤溶酶”与“纤维蛋白溶酶”、“纤维蛋白溶解酶”可互换使用，含义相同；“纤溶酶原”与“纤溶酶原”、“纤维蛋白溶解酶原”可互换使用，含义相同。
在本申请中，所述纤溶酶原“缺乏”的含义或活性为受试者体内纤溶酶原的含量比正常人低，低至足以影响所述受试者的正常生理功能；所述纤溶酶原“缺失”的含义或活性为受试者体内纤溶酶原的含量显著低于正常人，甚至活性或表达极微，只有通过外源提供才能维持正常生理功能。
本领域技术人员可以理解，本发明纤溶酶原的所有技术方案适用于纤溶酶，因此，本发明描述的技术方案涵盖了纤溶酶原和纤溶酶。
在循环过程中，纤溶酶原采用封闭的非活性构象，但当结合至血栓或细胞表面时，在纤溶酶原激活剂(plasminogen activator，PA)的介导下，其转变为呈开放性构象的活性纤溶酶。具有活性的纤溶酶可进一步将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体，进而溶解血栓。其中纤溶酶原的PAp结构域包含维持纤溶酶原处于非活性封闭构象的重要决定簇，而KR结构域则能够与存在于受体和底物上的赖氨酸残基结合。已知多种能够作为纤溶酶原激活剂的酶，包括：组织纤溶酶原激活剂(tPA)、尿激酶纤溶酶原激活剂(uPA)、激肽释放酶和凝血因子XII(哈格曼因子)等。
“纤溶酶原活性片段”是指在纤溶酶原蛋白中，能够与底物中的靶序列结合并发挥蛋白水解功能的活性片段。本发明涉及纤溶酶原的技术方案涵盖了用纤溶酶原活性片段代替纤溶酶原的技术方案。本发明所述的纤溶酶原活性片段为包含纤溶酶原的丝氨酸蛋白酶域的蛋白质，优选，本发明所述的纤溶酶原活性片段包含序列14、与序列14具有至少80％、90％、95％、96％、97％、98％、99％同源性的氨基酸序列的蛋白质。因此，本发明所述的纤溶酶原包括含有该纤溶酶原活性片段、并且仍然保持该纤溶酶原活性的蛋白。
目前，对于血液中纤溶酶原及其活性测定方法包括：对组织纤溶酶原激活剂活性的检测(t-PAA)、血浆组织纤溶酶原激活剂抗原的检测(t-PAAg)、对血浆组织纤溶酶原活性的检测(plgA)、血浆组织纤溶酶原抗原的检测(plgAg)、血浆组织纤溶酶原激活剂抑制物活性的检测、血浆组织纤溶酶原激活剂抑制物抗原的检测、血浆纤维蛋白溶酶-抗纤维蛋白溶酶复合物检测(PAP)。其中最常用的检测方法为发色底物法：向受检血浆中加链激酶(SK)和发色底物，受检血浆中的PLG在SK的作用下，转变成PLM，后者作用于发色底物，随后用分光光度计测定，吸光度增加与纤溶酶原活性成正比。此外也可采用免疫化学法、凝胶电泳、免疫比浊法、放射免疫扩散法等对血液中的纤溶酶原活性进行测定。
“直系同源物或直系同系物(ortholog)”指不同物种之间的同源物，既包括蛋白同源物也包括DNA同源物，也称为直向同源物、垂直同源物。其具体指不同物种中由同一祖先基因进化而来的蛋白或基因。本发明的纤 溶酶原包括人的天然纤溶酶原，还包括来源于不同物种的、具有纤溶酶原活性的纤溶酶原直系同源物或直系同系物。
“保守取代变体”是指其中一个给定的氨基酸残基改变但不改变蛋白质或酶的整体构象和功能，这包括但不限于以相似特性(如酸性，碱性，疏水性，等)的氨基酸取代亲本蛋白质中氨基酸序列中的氨基酸。具有类似性质的氨基酸是众所周知的。例如，精氨酸、组氨酸和赖氨酸是亲水性的碱性氨基酸并可以互换。同样，异亮氨酸是疏水氨基酸，则可被亮氨酸，蛋氨酸或缬氨酸替换。因此，相似功能的两个蛋白或氨基酸序列的相似性可能会不同。例如，基于MEGALIGN算法的70％至99％的相似度(同一性)。“保守取代变体”还包括通过BLAST或FASTA算法确定具有60％以上的氨基酸同一性的多肽或酶，若能达75％以上更好，最好能达85％以上，甚至达90％以上为最佳，并且与天然或亲本蛋白质或酶相比具有相同或基本相似的性质或功能。
“分离的”纤溶酶原是指从其天然环境分离和/或回收的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原会纯化(1)至大于90％、大于95％、或大于98％的纯度(按重量计)，如通过Lowry法所确定的，例如超过99％(按重量计)，(2)至足以通过使用旋转杯序列分析仪获得N端或内部氨基酸序列的至少15个残基的程度，或(3)至同质性，该同质性是通过使用考马斯蓝或银染在还原性或非还原性条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定的。分离的纤溶酶原也包括通过生物工程技术从重组细胞制备，并通过至少一个纯化步骤分离的纤溶酶原。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，指任何长度的氨基酸的聚合形式，其可以包括遗传编码的和非遗传编码的氨基酸，化学或生物化学修饰的或衍生化的氨基酸，和具有经修饰的肽主链的多肽。该术语包括融合蛋白，包括但不限于具有异源氨基酸序列的融合蛋白，具有异源和同源前导序列(具有或没有N端甲硫氨酸残基)的融合物；等等。
关于参照多肽序列的“氨基酸序列同一性百分数(％)”定义为在必要时引入缺口以实现最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分时，候选序列中与参照多肽序列中的氨基酸残基相同 的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式实现，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员能决定用于比对序列的适宜参数，包括对所比较序列全长实现最大对比需要的任何算法。然而，为了本发明的目的，氨基酸序列同一性百分数值是使用序列比较计算机程序ALIGN-2产生的。
在采用ALIGN-2来比较氨基酸序列的情况中，给定氨基酸序列A相对于给定氨基酸序列B的％氨基酸序列同一性(或者可表述为具有或包含相对于、与、或针对给定氨基酸序列B的某一％氨基酸序列同一性的给定氨基酸序列A)如下计算：
分数X/Y乘100
其中X是由序列比对程序ALIGN-2在该程序的A和B比对中评分为相同匹配的氨基酸残基的数目，且其中Y是B中的氨基酸残基的总数。应当领会，在氨基酸序列A的长度与氨基酸序列B的长度不相等的情况下，A相对于B的％氨基酸序列同一性会不等于B相对于A的％氨基酸序列同一性。除非另有明确说明，本文中使用的所有％氨基酸序列同一性值都是依照上一段所述，使用ALIGN-2计算机程序获得的。
如本文中使用的，术语“治疗”和“处理”指获得期望的药理和/或生理效果。所述效果可以是完全或部分预防疾病或其症状，和/或部分或完全治愈疾病和/或其症状，并且包括：(a)预防疾病在受试者体内发生，所述受试者可以具有疾病的素因，但是尚未诊断为具有疾病；(b)抑制疾病，即阻滞其形成；和(c)减轻疾病和/或其症状，即引起疾病和/或其症状消退。
术语“个体”、“受试者”和“患者”在本文中可互换使用，指哺乳动物，包括但不限于鼠(大鼠、小鼠)、非人灵长类、人、犬、猫、有蹄动物(例如马、牛、绵羊、猪、山羊)等。
“治疗有效量”或“有效量”指在对哺乳动物或其它受试者施用以治疗疾病时足以实现对疾病的所述预防和/或治疗的纤溶酶原的量。“治疗有效量”会根据所使用的纤溶酶原、要治疗的受试者的疾病和/或其症状的严重程度以及年龄、体重等而变化。
本发明纤溶酶原的制备
纤溶酶原可以从自然界分离并纯化用于进一步的治疗用途，也可以通过标准的化学肽合成技术来合成。当通过化学合成多肽时，可以经液相或固相进行合成。固相多肽合成(SPPS)(其中将序列的C末端氨基酸附接于不溶性支持物，接着序贯添加序列中剩余的氨基酸)是适合纤溶酶原化学合成的方法。各种形式的SPPS，诸如Fmoc和Boc可用于合成纤溶酶原。用于固相合成的技术描述于Barany和Solid-Phase Peptide Synthesis；第3-284页于The Peptides:Analysis,Synthesis,Biology.第2卷：Special Methods in Peptide Synthesis,Part A.,Merrifield,等J.Am.Chem.Soc.,85:2149-2156(1963)；Stewart等,Solid Phase Peptide Synthesis,2nd ed.Pierce Chem.Co.,Rockford,Ill.(1984)；和GanesanA.2006Mini Rev.Med Chem.6:3-10和Camarero JA等2005Protein Pept Lett.12:723-8中。简言之，用其上构建有肽链的功能性单元处理小的不溶性多孔珠。在偶联/去保护的重复循环后，将附接的固相游离N末端胺与单个受N保护的氨基酸单元偶联。然后，将此单元去保护，露出可以与别的氨基酸附接的新的N末端胺。肽保持固定在固相上，之后将其切掉。
可以使用标准重组方法来生产本发明的纤溶酶原。例如，将编码纤溶酶原的核酸插入表达载体中，使其与表达载体中的调控序列可操作连接。表达调控序列包括但不限于启动子(例如天然关联的或异源的启动子)、信号序列、增强子元件、和转录终止序列。表达调控可以是载体中的真核启动子系统，所述载体能够转化或转染真核宿主细胞(例如COS或CHO细胞)。一旦将载体掺入合适的宿主中，在适合于核苷酸序列的高水平表达及纤溶酶原的收集和纯化的条件下维持宿主。
合适的表达载体通常在宿主生物体中作为附加体或作为宿主染色体DNA的整合部分复制。通常，表达载体含有选择标志物(例如氨苄青霉素抗性、潮霉素抗性、四环素抗性、卡那霉素抗性或新霉素抗性)以有助于对外源用期望的DNA序列转化的那些细胞进行检测。
大肠杆菌(Escherichia coli)是可以用于克隆主题抗体编码多核苷酸的原核宿主细胞的例子。适合于使用的其它微生物宿主包括杆菌，诸如枯草芽孢杆菌(Bacillus subtilis)和其他肠杆菌科(enterobacteriaceae)，诸如沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、和各种假单胞菌属(Pseudomonas)物种。 在这些原核宿主中，也可以生成表达载体，其通常会含有与宿主细胞相容的表达控制序列(例如复制起点)。另外，会存在许多公知的启动子，诸如乳糖启动子系统，色氨酸(trp)启动子系统，beta-内酰胺酶启动子系统，或来自噬菌体λ的启动子系统。启动子通常会控制表达，任选在操纵基因序列的情况中，并且具有核糖体结合位点序列等，以启动并完成转录和翻译。
其他微生物，诸如酵母也可用于表达。酵母(例如酿酒酵母(S.cerevisiae))和毕赤酵母(Pichia)是合适的酵母宿主细胞的例子，其中合适的载体根据需要具有表达控制序列(例如启动子)、复制起点、终止序列等。典型的启动子包含3-磷酸甘油酸激酶和其它糖分解酶。诱导型酵母启动于特别包括来自醇脱氢酶、异细胞色素C、和负责麦芽糖和半乳糖利用的酶的启动子。
在微生物外，哺乳动物细胞(例如在体外细胞培养物中培养的哺乳动物细胞)也可以用于表达并生成本发明的抗-Tau抗体(例如编码主题抗-Tau抗体的多核苷酸)。参见Winnacker,From Genes to Clones,VCH Publishers,N.Y.,N.Y.(1987)。合适的哺乳动物宿主细胞包括CHO细胞系、各种Cos细胞系、HeLa细胞、骨髓瘤细胞系、和经转化的B细胞或杂交瘤。用于这些细胞的表达载体可以包含表达控制序列，如复制起点，启动子和增强子(Queen等,Immunol.Rev.89:49(1986))，以及必需的加工信息位点，诸如核糖体结合位点，RNA剪接位点，多聚腺苷酸化位点，和转录终止子序列。合适的表达控制序列的例子是白免疫球蛋白基因、SV40、腺病毒、牛乳头瘤病毒、巨细胞病毒等衍生的启动子。参见Co等,J.Immunol.148:1149(1992)。
一旦合成(化学或重组方式)，可以依照本领域的标准规程，包括硫酸铵沉淀，亲和柱，柱层析，高效液相层析(HPLC)，凝胶电泳等来纯化本发明所述的纤溶酶原。该纤溶酶原是基本上纯的，例如至少约80％至85％纯的，至少约85％至90％纯的，至少约90％至95％纯的，或98％至99％纯的或更纯的，例如不含污染物，所述污染物如细胞碎片，除主题抗体以外的大分子，等等。
药物配制剂
可以通过将具有所需纯度的纤溶酶原与可选的药用载体，赋形剂，或稳定剂(Remington's Pharmaceutical Sciences,16版，Osol,A.ed.(1980))混合形成 冻干制剂或水溶液制备治疗配制剂。可接受的载体、赋形剂、稳定剂在所用剂量及浓度下对受者无毒性，并包括缓冲剂例如磷酸盐，柠檬酸盐及其它有机酸；抗氧化剂包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化己烷双胺；氯化苄烷铵(benzalkonium chloride)，苯索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如甲基或丙基对羟基苯甲酸酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；间甲酚)；低分子量多肽(少于约10个残基)；蛋白质如血清白蛋白，明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸，谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖，二糖及其它碳水化合物包括葡萄糖、甘露糖、或糊精；螯合剂如EDTA；糖类如蔗糖、甘露醇、岩藻糖或山梨醇；成盐反离子如钠；金属复合物(例如锌-蛋白复合物)；和/或非离子表面活性剂，例如TWEENTM，PLURONICSTM或聚乙二醇(PEG)。优选冻干的抗-VEGF抗体配制剂在WO 97/04801中描述，其包含在本文中作为参考。
本发明的配制剂也可含有需治疗的具体病症所需的一种以上的活性化合物，优选活性互补并且相互之间没有副作用的那些。例如，抗高血压的药物，抗心律失常的药物，治疗糖尿病的药物等。
本发明的纤溶酶原可包裹在通过诸如凝聚技术或界面聚合而制备的微胶囊中，例如，可置入在胶质药物传送系统(例如，脂质体，白蛋白微球，微乳剂，纳米颗粒和纳米胶囊)中或置入粗滴乳状液中的羟甲基纤维素或凝胶-微胶囊和聚-(甲基丙烯酸甲酯)微胶囊中。这些技术公开于Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed.(1980)。
用于体内给药的本发明的纤溶酶原必需是无菌的。这可以通过在冷冻干燥和重新配制之前或之后通过除菌滤膜过滤而轻易实现。
本发明的纤溶酶原可制备缓释制剂。缓释制剂的适当实例包括具有一定形状且含有糖蛋白的固体疏水聚合物半通透基质，例如膜或微胶囊。缓释基质实例包括聚酯、水凝胶(如聚(2-羟基乙基-异丁烯酸酯)(Langer等，J.Biomed.Mater.Res.,15:167-277(1981)；Langer,Chem.Tech.,12:98-105(1982))或聚(乙烯醇)，聚交酯(美国专利3773919,EP 58,481)，L-谷氨酸与γ乙基-L-谷氨酸的共聚物(Sidman，等，Biopolymers 22:547(1983))，不可降解的乙烯－乙烯乙酸酯(ethylene-vinyl acetate)(Langer，等，出处同上)，或可降解的乳酸-羟基 乙酸共聚物如Lupron DepotTM(由乳酸-羟基乙酸共聚物和亮氨酰脯氨酸(leuprolide)乙酸酯组成的可注射的微球体)，以及聚D-(-)-3-羟丁酸。聚合物如乙烯-乙酸乙烯酯和乳酸-羟基乙酸能持续释放分子100天以上，而一些水凝胶释放蛋白的时间却较短。可以根据相关机理来设计使蛋白稳定的合理策略。例如，如果发现凝聚的机理是通过硫代二硫键互换而形成分子间S-S键，则可通过修饰巯基残基、从酸性溶液中冻干、控制湿度、采用合适的添加剂、和开发特定的聚合物基质组合物来实现稳定。
给药和剂量
可以通过不同方式，例如通过静脉内，腹膜内，皮下，颅内，鞘内，动脉内(例如经由颈动脉)，肌内来实现本发明药物组合物的施用。
用于胃肠外施用的制备物包括无菌水性或非水性溶液、悬浮液和乳剂。非水性溶剂的例子是丙二醇、聚乙二醇、植物油如橄榄油，和可注射有机酯，如油酸乙酯。水性载体包括水、醇性/水性溶液、乳剂或悬浮液，包括盐水和缓冲介质。胃肠外媒介物包含氯化钠溶液、林格氏右旋糖、右旋糖和氯化钠、或固定油。静脉内媒介物包含液体和营养补充物、电解质补充物，等等。也可以存在防腐剂和其他添加剂，诸如例如，抗微生物剂、抗氧化剂、螯合剂、和惰性气体，等等。
医务人员会基于各种临床因素确定剂量方案。如医学领域中公知的，任一患者的剂量取决于多种因素，包括患者的体型、体表面积、年龄、要施用的具体化合物、性别、施用次数和路径、总体健康、和同时施用的其它药物。本发明包含纤溶酶原的药物组合物的剂量范围可以例如为每天约0.0001至2000mg/kg，或约0.001至500mg/kg(例如0.02mg/kg，0.25mg/kg，0.5mg/kg，0.75mg/kg，10mg/kg，50mg/kg等等)受试者体重。例如，剂量可以是1mg/kg体重或50mg/kg体重或在1-50mg/kg的范围，或至少1mg/kg。高于或低于此例示性范围的剂量也涵盖在内，特别是考虑到上述的因素。上述范围中的中间剂量也包含在本发明的范围内。受试者可以每天、隔天、每周或根据通过经验分析确定的任何其它日程表施用此类剂量。例示性的剂量日程表包括连续几天1-10mg/kg。在本发明的药物施用过程中需要实时评估治疗效果和安全性。
制品或药盒
本发明的一个实施方案涉及一种制品或药盒，其包含可用于治疗骨质疏松及其相关病症的本发明纤溶酶原或纤溶酶。所述制品优选包括一个容器，标签或包装插页。适当的容器有瓶子，小瓶，注射器等。容器可由各种材料如玻璃或塑料制成。所述容器含有组合物，所述组合物可有效治疗本发明的疾病或病症并具有无菌入口(例如所述容器可为静脉内溶液包或小瓶，其含有可被皮下注射针穿透的塞子的)。所述组合物中至少一种活性剂为纤溶酶原/纤溶酶。所述容器上或所附的标签说明所述组合物用于治疗本发明所述骨质疏松及其相关病症。所述制品可进一步包含含有可药用缓冲液的第二容器，诸如磷酸盐缓冲的盐水，林格氏溶液以及葡萄糖溶液。其可进一步包含从商业和使用者角度来看所需的其它物质，包括其它缓冲液，稀释剂，过滤物，针和注射器。此外，所述制品包含带有使用说明的包装插页，包括例如指示所述组合物的使用者将纤溶酶原组合物以及治疗伴随的疾病的其它药物给药患者。
治疗效力和治疗安全性
本发明的一个实施方案涉及使用纤溶酶原治疗受试者后，对治疗效力和治疗安全性的判断。常用的骨质疏松症治疗效果监测与评估内容包括随访(不良反应、规范服药、基础措施以及骨折风险因子再评估等)，新发骨折评估(临床骨折、身高降低和影像学检查)、骨密度(bone mineral density，BMD)测量和骨转换生化标志物(bone turnover markers，BTM)检测，以及基于这些数据的综合再评估等。其中BMD和骨量是目前应用最广泛的疗效监测和评估方法例如，可以通过双能X线骨密度仪(dual energy X-ray absorptiometry，DXA)、定量CT(quantitative computed tomography，QCT)、单光子吸收测定法(SPA)、或超声波测定法来测量BMD。治疗开始后可每年检测1次BMD，在BMD达到稳定后可以适当延长间隔，例如2年监测1次。针对BTM，目前在血清学指标中较多使用的骨形成指标是血清1型原胶原N端前肽(procollagen type 1n-terminal propeptide，PINP)，骨吸收指标是血清1型原胶原C末端肽(serum C-terminal telopeptide，S-CTX)。可根据研究进展， 适时调整更合理的检测指标。应在开始治疗前检测基线值，应用促形成药物治疗后3个月、应用抑制吸收药物治疗后3～6个月时进行检测。BTM能够提供骨骼的动态信息，在作用和功能上独立于BMD，同时也与BMD成为互为补充的监测手段，二者结合起来具有更高的临床价值。一般地，如果治疗后BMD上升或稳定，BTM有预期变化，同时治疗期间无骨折发生，可认为治疗反应良好。此外，本发明还涉及使用纤溶酶原及其变体对受试者进行治疗过程中和治疗后，所述该治疗方案安全性的判断，包括但不限于对药物在受试者体内的血清半衰期、治疗半衰期、半数中毒量(TD50)、半数致死量(LD50)进行统计，或对在治疗过程中或治疗后发生的各种不良事件如致敏反应进行观察。
附图简述
图1显示15周龄野生型和纤溶酶原缺陷型小鼠的膝关节Safranin O染色代表性图片。A为野生型小鼠，B为纤溶酶原缺陷小鼠。与野生型小鼠相比，纤溶酶原缺陷型小鼠展现了广泛的骨质减少和骨髓细胞增加。
图2显示15周龄纤溶酶原缺陷型以及野生型小鼠血钙检测结果。结果显示，纤溶酶原缺陷型(Ko)小鼠血钙水平显著高于野生型小鼠(Wt)，且统计差异显著(*表示P<0.05)。说明纤溶酶原在维持正常钙代谢上发挥着重要作用。
图3显示纤溶酶原缺陷型(Plg-/-)小鼠给予纤溶酶原30天后膝关节H&E染色观察结果。A、C为给溶媒PBS对照组，B、D为给纤溶酶原组。结果显示，给溶媒PBS对照组，生长板(箭头标识)排列紊乱，部分骨髓腔内骨髓消失(三角形标识)；给纤溶酶原组，生长板(箭头标识)排列整齐。说明纤溶酶原能够促进Plg-/-小鼠膝关节生长板的正常生长。
图4显示Plg-/-小鼠给予纤溶酶原30天后膝关节关节软骨碱性磷酸酶染色观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给溶媒PBS对照组关节软骨表面只有极少量的碱性磷酸酶着色，而给纤溶酶原组关节软骨表面却有较多的呈深红色的碱性磷酸酶着色(箭头标识)。说明给纤溶酶原组关节软骨表面的碱性磷酸酶活性明显高于PBS对照组，即纤溶酶原促使膝关节关节软骨的成骨细胞活性明显增加。
图5显示Plg-/-小鼠给予纤溶酶原30天后膝关节生长板碱性磷酸酶染色观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给溶媒PBS对照组在生长板成骨细胞活性处有碱性磷酸酶着色(箭头标识)，呈浅红色；给纤溶酶原组在生长板处有较多碱性磷酸酶着色，且呈深红色。说明给纤溶酶原之后，可以促进膝关节生长板的成骨细胞活性的增加。
图6显示0.5μg/kg维生素D衰老模型C57小鼠给予纤溶酶原28天后血清碱性磷酸酶检测结果。结果显示，给纤溶酶原组小鼠血清碱性磷酸酶活性明显高于给溶媒PBS对照组小鼠，且统计差异显著(*表示P<0.05)，并且与给溶媒PBS对照组相比给纤溶酶原组小鼠血清碱性磷酸酶活性更加接近空白对照组小鼠。这提示，纤溶酶原组能够显著促进维生素D衰老模型小鼠成骨细胞活性的增加。
图7显示1μg/kg维生素D衰老模型C57小鼠给予纤溶酶原28天膝关节生长板碱性磷酸酶染色代表性图片。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。结果显示，给溶媒PBS对照组膝关节生长板碱性磷酸酶阳性着色(箭头标识)明显少于空白对照小鼠；给纤溶酶原组膝关节生长板碱性磷酸酶阳性着色明显高于给溶媒PBS对照组小鼠。说明纤溶酶原能够改善维生素D诱导的衰老模型小鼠膝关节生长板成骨细胞的活性
图8不同周龄段Plg-/-和Plg+/+小鼠头盖骨Micro CT骨密度检测结果。A为皮质骨密度，B为头盖骨总骨密度。结果显示，随着周龄的增大，Plg+/+小鼠皮质骨密度和总骨密度有逐渐增加的趋势，而Plg-/-小鼠头盖骨皮质骨密度和总骨密度逐渐降低。而两个品系的小鼠骨密度在20-21周龄就有极其显著差异，在29-30周龄时差异更加显著。说明纤溶酶原在头盖骨骨密度调节中发挥重要作用，与骨质疏松密切相关。
图9 20-21周龄周龄段Plg/-和Plg+/+小鼠头盖骨Micro CT骨矿物含量检测结果。结果显示，20-21周龄Plg+/+小鼠皮质骨和总骨骨矿物含量明显高于Plg/-小鼠，且统计差异显著。说明纤溶酶原在头盖骨矿物含量调节上发挥重要作用，与骨质疏松密切相关。
图10不同周龄段Plg-/-和Plg+/+小鼠股骨Micro CT骨密度检测结果。A为皮质骨密度，B为松质骨密度，C为小梁骨密度，D为总骨密度。结果显示，在12-30周龄期间随着周龄增大Plg+/+小鼠股骨密度逐渐增加，而Plg-/-小 鼠股骨皮质骨密度、松质骨密度、小梁骨密度以及总骨密度逐渐减小。在此期间，Plg+/+小鼠股骨密度高于Plg-/-小鼠，在20周龄时两个品系小鼠骨密度差异显著，并且随着周龄增大，二者差异越来越显著。说明纤溶酶原参与股骨密度调节，并在一定时期发挥重要作用。
图11不同周龄段Plg-/-和Plg+/+小鼠股骨Micro CT骨矿物含量检测结果。A为皮质骨矿物含量，B为松质骨矿物含量，C为小梁骨矿物含量，D为总骨矿物含量。结果显示，在12-30周龄期间随着周龄增大Plg+/+小鼠股骨不同部分矿物含量变化不大或逐步增加，而Plg-/-小鼠股骨松质骨和小梁骨矿物含量逐渐减小。在此期间，Plg+/+小鼠股骨矿物含量高于Plg-/-小鼠，在20周龄时两个品系小鼠股骨皮质骨、小梁骨和总骨骨矿物含量差异显著，并且随着周龄增大，二者骨矿物含量差异越来越显著。说明纤溶酶原参与股骨矿物质代谢调节，并在一定时期发挥重要作用。
图12不同周龄段Plg-/-和Plg+/+小鼠腰椎骨Micro CT骨密度检测结果。A为皮质骨密度，B为松质骨密度，C为小梁骨密度，D为总骨密度。结果显示，在12-30周龄期间随着周龄增大Plg+/+小鼠腰椎骨骨密度逐渐增大，而Plg-/-小鼠腰椎骨皮质骨密度、松质骨密度、小梁骨密度以及总骨密度逐渐减小。在此期间，Plg+/+小鼠骨密度高于Plg-/-小鼠，在12周龄时两个品系小鼠腰椎骨骨密度差异显著，并且随着周龄的增大，二者差异越来越显著。说明纤溶酶原参与腰椎骨密度调节，并在一定时期发挥重要作用。
图13不同周龄段Plg-/-和Plg+/+小鼠腰椎骨Micro CT骨矿物含量检测结果。A为皮质骨矿物含量，B为松质骨矿物含量，C为小梁骨矿物含量，D为总骨矿物含量。结果显示，在12-30周龄期间随着周龄增大Plg+/+小鼠腰椎骨不同部分矿物含量变化不大，而Plg-/-小鼠腰椎骨皮质骨、松质骨、小梁骨以及总骨矿物含量逐渐减小。在此期间，Plg+/+小鼠腰椎骨矿物含量高于Plg- /-小鼠，在20周龄时两个品系小鼠腰椎骨松质骨和皮质骨区域矿物含量差异显著，并且随着周龄增大，二者差异越来越显著。说明纤溶酶原参与腰椎骨矿物质代谢调节，并在一定时期发挥重要作用。
图14不同周龄段Plg-/-和Plg+/+小鼠血清碱性磷酸酶检测结果。结果显示，Plg+/+小鼠血清碱性磷酸酶活性在12-30周龄时虽有波动但变化并不显著，而Plg-/-小鼠血清碱性磷酸酶的活性随着周龄的增大逐渐的降低；Plg+/+ 小鼠血清碱性磷酸酶的活性明显高于Plg-/-小鼠，两个品系小鼠血清碱性磷酸酶活性在12周龄时就出现明显的差异，并且随着周龄的增大，差异越来越显著。该结果提示纤溶酶原可能促进成骨细胞的活性，促进骨重建。
图15显示ApoE动脉粥样硬化模型小鼠给予纤溶酶原30天后血钙检测结果。结果显示，给纤溶酶原组小鼠血钙浓度要明显的低于给溶媒PBS对照组，且统计差异显著(*表示P<0.05)。说明纤溶酶原能够降低ApoE动脉粥样硬化模型小鼠血钙的含量。
图16显示ApoE动脉粥样硬化模型小鼠给予纤溶酶原30天后主动脉窦茜素红染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给纤溶酶原组小鼠主动脉窦钙沉积明显少于给溶媒PBS对照组。说明纤溶酶原能够改善动脉粥样硬化中主动脉窦钙化。
图17动脉粥样硬化模型小鼠给予纤溶酶原后股骨密度检测结果。A为皮质骨密度，B为松质骨密度，C为小梁骨密度，D为总骨密度。结果显示，给药10天后给纤溶酶原组小鼠股骨密度明显高于给溶媒PBS对照组，且松质骨密度和总骨密度差异显著(*表示P<0.05)；给药30天后给纤溶酶原组小鼠松质骨密度、小梁骨密度和总骨密度相较于给溶媒PBS对照组显著升高，且统计差异显著(*表示P<0.05)。皮质骨密度两组无明显差异。说明纤溶酶原能够促进动脉粥样硬化模型小鼠骨密度升高，改善动脉粥样硬化所致的骨质疏松。
图18动脉粥样硬化模型小鼠给予纤溶酶原后膝关节H&E染色代表性图片。A-C为给溶媒PBS对照组，D-F为给纤溶酶原组。结果显示，给溶媒PBS对照组软骨表面轻度纤维化(细箭头标识)，小梁骨(三角标识)明显变细，粗细不均匀，软骨组织(星形标识)排列紊乱，生长板(粗箭头标识)层次紊乱，软骨细胞轻度减少，潮线基本清晰；给纤溶酶原组关节软骨表面基本正常，潮线清晰，骨小梁粗细均匀，生长板结构清晰，层次规则而可分。说明纤溶酶原能够改善ApoE动脉粥样硬化模型小鼠膝关节的状况。
图19给予纤溶酶原对C57卵巢切除及注射地塞米松诱导骨质疏松模型小鼠体重影响。结果显示，给溶媒PBS对照组小鼠体重明显轻于正常对照组，而给纤溶酶原组体重明显高于给溶媒PBS对照组，且统计差异显著 (P<0.05)。说明纤溶酶原能够显著促进卵巢切除及注射地塞米松诱导骨质疏松模型小鼠体重恢复。
图20给予纤溶酶原后C57卵巢切除及注射地塞米松诱导骨质疏松模型小鼠股骨Micro CT扫描测定结果。A为骨体积测量结果，B为骨矿物含量测量结果。结果显示，给纤溶酶原组小鼠股骨的松质骨、小梁骨以及总骨体积和骨矿物含量均大于给溶媒PBS对照组，且均统计差异显著(*表示P<0.05)。说明纤溶酶原能够促进骨质疏松模型小鼠股骨矿物质的沉积、骨体积增加，改善骨质疏松。
图21给予纤溶酶原C57卵巢切除及注射地塞米松诱导骨质疏松模型小鼠股骨Micro CT扫描测定结果。
图21A为股骨骨密度测量结果。结果显示，给溶媒PBS对照组小鼠股骨皮质骨、松质骨、小梁骨和总骨骨密度均小于正常对照组；而给纤溶酶原组小鼠各部分骨密度大于给溶媒PBS对照组。趋势明确，但由于小鼠数量少，造成统计差异只是接近显著。可预期增加小鼠数量会出现统计学差异。
图21B为股骨骨矿物含量测量结果。结果显示，给溶媒PBS对照组小鼠股骨各部分骨矿物含量均小于正常对照组；而给纤溶酶原组小鼠各部分骨矿物含量均大于给溶媒PBS对照组。趋势明确，但由于小鼠数量少，造成统计差异只是接近显著。可预期增加小鼠数量会出现统计学差异。
图21C为小梁骨体积测量结果。结果显示，给溶媒PBS对照组小鼠股骨的小梁骨体积小于正常对照组；而给纤溶酶原组小鼠股骨小梁骨体积大于给溶媒PBS对照组。趋势明确，但由于小鼠数量少，造成统计差异只是接近显著。可预期增加小鼠数量会出现统计学差异。
综上所述，纤溶酶原能够明显改善骨质疏松，促进股骨各部分骨密度和骨量的增加，并且对小梁骨改善作用尤为明显。
图22给予纤溶酶原C57卵巢切除及注射地塞米松诱导骨质疏松模型小鼠膝关节H&E染色和Safrain O染色代表性图片。A、C为给溶媒PBS组，B、D为给纤溶酶原组。结果显示，给溶媒PBS组骨小梁(箭头标识)明显变细，断裂，出现较大面积的无骨小梁骨髓腔，髓腔增大，骨小梁的连接中断，生长板下骨细胞轻度减少(三角形标识)；给纤溶酶原组骨小梁部分变细， 较之于PBS组，骨小梁连续性较好，较粗，没有较大面积的无骨小梁区域，软骨组织层次结构也较规则。说明给予纤溶酶原能明显改善骨质疏松模型小鼠膝关节的状况。
图23给予纤溶酶原C57卵巢切除及注射地塞米松诱导骨质疏松模型小鼠膝关节碱性磷酸酶染色代表性图片。A、C为给溶媒PBS对照组，B、D为给纤溶酶原组。结果显示，给溶媒PBS对照组小鼠膝关节软骨组织(细箭头标识)和生长板(粗箭头标识)碱性磷酸酶着色明显少于给纤溶酶原组。说明纤溶酶原促进骨质疏松模型小鼠膝关节成骨细胞活性增加。
图24给予纤溶酶原后Plg+/+卵巢切除诱导的骨质疏松模型小鼠血清钙检测结果。结果显示，给纤溶酶原组小鼠血清钙浓度明显低于给溶媒PBS对照组，且统计差异显著(*表示P<0.05)。说明纤溶酶原能够显著的降低卵巢切除骨质疏松模型小鼠血钙的浓度。
图25给予纤溶酶原后Plg+/+卵巢切除诱导的骨质疏松模型小鼠血清磷检测结果。结果显示，给纤溶酶原组小鼠血清磷浓度明显高于给溶媒PBS对照组，且统计差异显著(*表示P<0.05)。说明纤溶酶原能够显著的升高卵巢切除骨质疏松模型小鼠血磷的浓度。
图26给予纤溶酶原3％胆固醇高脂血症模型小鼠膝关节碱性磷酸酶染色结果。A、C为给溶媒PBS对照组，B、D为给纤溶酶原组，E为定量分析结果。结果显示，给纤溶酶原组小鼠膝关节碱性磷酸酶着色(箭头标识)明显多于给溶媒PBS对照组，且统计差异显著(*表示P<0.05)。说明纤溶酶原显著增加3％胆固醇高脂血症模型小鼠膝关节成骨细胞活性。
图27给予纤溶酶原C57卵巢切除及注射地塞米松诱导骨质疏松模型小鼠膝关节H&E染色和Safrain O染色代表性图片。A、C为给溶媒PBS组，B、D为给纤溶酶原组。结果显示，给溶媒PBS对照组小鼠膝关节骨小梁(箭头标识)明显变细，断裂，出现较大面积的无骨小梁骨髓腔，骨小梁的连接中断，关节表面部分纤维化，生长板下成骨区成骨组织明显减少(三角形标识)；给纤溶酶原组骨小梁部分变细，较之于PBS对照组，骨小梁连续性较好，没有较严重的断裂，没有较大面积的无骨小梁区域，软骨组织层次结构也较规则，潮线清晰。说明给予纤溶酶原能明显改善骨质疏松模型小鼠膝关节的状况。
实施例
材料与方法:
动物：C57小鼠和Plg+/+以及Plg-/-小鼠(Jackson Lab)用于相关实验。动物饲养于符合国标的实验动物使用环境。
试剂：维生素D(Sigma Aldrich，货号D1530)，玉米油(Sigma Aldrich，货号C8267，)，低钙特殊饲料(0.2％钙，1％磷酸盐，2000U维生素D3/kg，南通特洛菲饲料科技有限公司，15kg)，钙含量测定试剂盒(南京建成生物工程研究所，货号C004-2)，人纤溶酶原(10mg/ml,纯化自健康血浆捐献者)。
Aloka Micro CT专用于小鼠、大鼠形态观察，采用最新的第三代X射线测量，短时间内即可获得高质量断层扫描图像。可用于骨测量(骨密度、骨矿物质含量、骨体积、骨微结构等)、体脂肪率测量、内脏、皮下脂肪的辨别及测量、同步摄影等。骨测量以小鼠股骨、头盖骨或腰椎骨为检测对象。小鼠处死后取材股骨、头盖骨和腰椎骨于4％多聚甲醛固定，采用Micro CT(Aloka,日本HITACHI公司生产)，对骨进行测定。
实施例1纤溶酶原缺乏与骨质疏松密切相关
15周龄野生型和纤溶酶原缺陷型(Plg-/-)小鼠各5只。取膝关节于4％多聚甲醛固定24小时，然后10％EDTA中脱钙三周，梯度蔗糖溶液洗涤，以上操作需在4℃条件下进行。然后石蜡包埋，8μm切片行Safranin O染色。切片在200倍光学显微镜下观察。
结果显示，与野生型小鼠(图1A)相比，Plg-/-小鼠(图1B)展现了广泛的骨质减少和骨髓细胞增加。
实施例2野生小鼠和纤溶酶原缺陷型小鼠的钙流失对比
15周龄野生型(wt)和纤溶酶原缺陷型(ko)小鼠各5只。两组小鼠摘除眼球取血，检测血钙浓度。在正常情况下，体内钙平衡受到非常精密的调节。然而，在骨质疏松的情况下，钙流失是骨质疏松的一个关键标志。我们研究在野生型和Plg-/-小鼠中钙的水平发现，Plg-/-(Ko)小鼠在15周龄时血钙水平显著高于野生型小鼠，且统计差异显著(*表示P<0.05)(图2)。
实施例3纤溶酶原对Plg-/-小鼠膝组织结构的保护作用
20周龄的Plg-/-小鼠8只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组各4只。实验第1天称重分组并开始给纤溶酶原或溶媒PBS，给纤溶酶原组按1mg/0.1mL/只/天经尾静脉注射给予纤溶酶原，给溶媒PBS对照组尾静脉注射给予相同体积的PBS。连续给药30天，第31天处死小鼠，取膝关节于固定液中4℃固定24小时。固定液配方:2％多聚甲醛，0.075mol/L赖氨酸，0.01mol/L过碘酸钠。固定后4℃PBS洗液梯度洗涤各12小时，然后置于4℃脱钙液中脱钙2周，每5天换一次脱钙液。脱钙完成后4℃PBS洗液梯度洗涤12小时，膝关节经酒精梯度脱水、二甲苯透明浸蜡后进行石蜡包埋。切片厚度5um，切片脱蜡复水，并用苏木素和伊红染色(H&E染色)，1％盐酸酒精分化，氨水返蓝，并酒精梯度脱水，二甲苯透明，中性树胶封片，切片在200倍光学显微镜下观察。
结果显示，给溶媒PBS对照组(图3A、C)，生长板(箭头标识)排列紊乱，部分骨髓腔内骨髓消失(三角形标识)；给纤溶酶原组(图3B、D)，生长板排列整齐。说明纤溶酶原能促进Plg-/-小鼠膝关节生长板的生长。
实施例4纤溶酶原促进Plg-/-小鼠膝关节关节软骨表面成骨细胞活性的增加
20周龄的Plg-/-小鼠8只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组各4只。实验第1天称重分组并开始给纤溶酶原或溶媒PBS，给纤溶酶原组按1mg/0.1mL/只/天经尾静脉注射给予纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。连续给药30天，第31处死小鼠，取股骨与固定液中4℃固定24小时。固定液配方:2％多聚甲醛，0.075mol/L赖氨酸，0.01mol/L过碘酸钠。固定后4℃PBS洗液梯度洗涤各12小时，然后置于4℃脱钙液中脱钙2周，每5天换一次脱钙液。脱钙完成后4℃PBS洗液梯度洗涤12小时，膝关节经酒精梯度脱水和二甲苯透明后进行石蜡包埋。切片5um，脱蜡复水，氯化镁缓冲液4℃孵育过夜。碱性磷酸酶底物溶液室温孵育1小时，苏木素复染2分钟。流水冲洗5分钟，60℃烤30分钟，中性树胶封片，切片在200倍光学显微镜下观察。
碱性磷酸酶(alkaline phosphatase，ALP)是成骨细胞早期分化的标志[33]。结果显示，给溶媒PBS对照组(图4A)关节软骨表面只有极少量的碱性磷酸酶着色(箭头标识)，而给纤溶酶原组(图4B)关节软骨表面却有较多 的呈深红色的碱性磷酸酶着色。说明给纤溶酶原组关节软骨表面的碱性磷酸酶活性明显高于对照组，即纤溶酶原促使膝关节关节软骨的成骨细胞活性明显增加。
实施例5纤溶酶原促进Plg-/-小鼠膝关节生长板成骨细胞活性的增加
20周龄的Plg-/-小鼠8只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组各4只。实验第1天称重分组并开始给纤溶酶原或溶媒PBS，给纤溶酶原组按1mg/0.1mL/只/天经尾静脉注射给予纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。连续给药30天，第31处死小鼠，取股骨与固定液中4℃固定24小时。固定液配方:2％多聚甲醛，0.075mol/L赖氨酸，0.01mol/L过碘酸钠。固定后4℃PBS洗液梯度洗涤各12小时，然后置于4℃脱钙液中脱钙2周，每5天换一次脱钙液。脱钙完成后4℃PBS洗液梯度洗涤12小时，股骨经酒精梯度脱水和二甲苯透明后进行石蜡包埋。切片5um，脱蜡复水，氯化镁缓冲液4℃孵育过夜。碱性磷酸酶底物溶液室温孵育1小时，苏木素复染2分钟。流水冲洗5分钟，60℃烤30分钟，中性树胶封片，在显微镜下200倍下观察拍照。
结果显示，给溶媒PBS对照组(图5A)在生长板成骨细胞活性处有碱性磷酸酶着色(箭头标识)，呈浅红色；给纤溶酶原组(图5B)在生长板处有较多碱性磷酸酶着色，呈深红色。说明给纤溶酶原之后，可以促进膝关节生长板的成骨细胞活性的增加。说明给纤溶酶原之后，可以促进膝关节生长板的成骨细胞活性的增加。
实施例6纤溶酶原改善维生素D诱导的衰老模型小鼠血清碱性磷酸酶活性
取5-6周龄的雄性C57小鼠25只，称重后随机分为3组，空白对照组5只，给纤溶酶原组10只和给溶媒PBS对照组10只。空白对照组小鼠每天腹腔注射50μl玉米油；给纤溶酶原组和给溶媒PBS对照组小鼠按照0.5μg/kg/天腹腔注射维生素D(Sigma Aldrich)，诱导衰老[34,35]。与此同时小鼠开始给药，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，空白对照组小鼠不做给药处理，连续造模给药28天。给药期间空白对照组小鼠饲喂低钙饲料，给纤溶酶原组和给溶媒PBS对照组小鼠饲喂低钙饲料。开始造模给药定为第1天，第29天摘除眼球 取血，离心获得上清，用以检测血清碱性磷酸酶(alkaline phosphatase，ALP)的活性。
结果显示，给纤溶酶原组小鼠血清碱性磷酸酶活性明显高于给溶媒PBS对照组小鼠，且统计差异显著，并且与给溶媒PBS对照组相比给纤溶酶原组小鼠血清碱性磷酸酶活性更加接近空白对照组小鼠(图6)。
血清ALP是一种同工酶糖蛋白，血清ALP主要来源于肝脏和骨骼，其中来源于骨骼的ALP占40％～75％。ALP活性测定主要用于诊断肝胆和骨骼系统疾病。临床上除肝脏疾患、妊娠等因素外，血清ALP还可反映成骨情况。骨代谢旺盛时成骨细胞活跃，ALP分泌量增加，存在于成骨细胞周围及其表面，极易释放入血中，使血清ALP活性上升，因此，血清ALP是骨重建活跃性改变的标志之一[36]。
本研究中，给纤溶酶原组小鼠血清碱性磷酸酶活性明显高于给溶媒PBS对照组小鼠，且统计差异显著。这提示，纤溶酶原组能够显著促进维生素D衰老模型小鼠成骨细胞活性的增加。
实施例7纤溶酶原促进维生素D诱导的衰老模型小鼠膝关节生长板碱性磷酸酶活性增加
取5-6周龄的雄性C57小鼠15只，称重后随机分为3组，空白对照组、给纤溶酶原组和给溶媒PBS对照组，每组各5只。空白对照组小鼠每天腹腔注射50μl玉米油；给纤溶酶原组和给溶媒PBS对照组小鼠按照1μg/kg/天腹腔注射维生素D(Sigma Aldrich)，诱导衰老[34,35]。与此同时小鼠开始给药，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，空白对照组小鼠不做给药处理，连续造模给药28天。给药期间所有小鼠饲喂低钙饲料(南通特诺菲)。开始造模给药定为第1天，第29天处死小鼠取材膝关节于固定液中固定24小时。固定液配方:2％多聚甲醛，0.075mol/L赖氨酸，0.01mol/L过碘酸钠。固定后4℃PBS洗液梯度洗涤各12小时，然后置于4℃脱钙液中脱钙2周，每5天换一次脱钙液。脱钙完成后4℃PBS洗液梯度洗涤12小时，膝关节经酒精梯度脱水和二甲苯透明后进行石蜡包埋。切片5um，脱蜡复水，氯化镁缓冲液4℃孵育过夜。碱性磷酸酶底物溶液室温孵育1小时，苏木素复染2分钟。流水冲洗5分钟，60℃烤30分钟，中性树胶封片，切片在200倍光学显微镜下观察。
结果显示，给溶媒PBS对照组(图7B)膝关节生长板碱性磷酸酶阳性着色(箭头标识)明显少于空白对照小鼠(图7A)；给纤溶酶原组(图7C)膝关节生长板碱性磷酸酶阳性着色明显高于给溶媒PBS对照组小鼠。说明纤溶酶原能够改善维生素D诱导的衰老模型小鼠膝关节生长板成骨细胞的活性。
实施例8纤溶酶原对头盖骨密度的影响
取12-13、20-21、29-30周龄的Plg+/+小鼠和Plg-/-小鼠各5只，各组小鼠体重基本相同。小鼠在实验过程中都饲以同样的食物和水。取头盖骨于4％多聚甲醛固定24小时，进行Micro CT扫描，测定骨密度。
结果显示，随着周龄的增大，而Plg+/+小鼠皮质骨密度(图8A)和总骨密度(图8B)有逐渐增加的趋势，而Plg-/-小鼠头盖骨皮质骨密度和总骨密度逐渐降低。两个品系的小鼠骨密度在20-21周龄有极其显著差异，在29-30周龄时差异更加显著。说明纤溶酶原参与骨矿物质代谢调节，并在一定时期发挥重要作用。
骨质疏松是以骨量减少、骨的微观结构退化为特征，致使骨脆性增加、易于骨折的一种全身性骨骼疾病。WHO建议采用骨密度(bone mineral density，BMD)测量结果诊断骨质疏松症[37,38]。上述实验结果说明，说明纤溶酶原参与骨矿物质代谢调节，并在一定时期发挥重要作用。
实施例9纤溶酶原对头盖骨矿物含量的影响
取20-21周龄的Plg+/+小鼠和Plg-/-小鼠各5只，小鼠体重基本相同。小鼠在实验过程中都饲以同样的食物和水。取头盖骨于4％多聚甲醛固定24小时，进行Micro CT扫描，测定骨矿物含量。
结果显示，结果显示，20-21周龄Plg+/+小鼠皮质骨和总骨骨矿物含量明显高于Plg/-小鼠，且统计差异极其显著或显著。说明纤溶酶原在头盖骨矿物含量调节上发挥重要作用，与骨质疏松密切相关。
实施例10纤溶酶原缺乏小鼠股骨密度降低
取12-13、20-21、29-30周龄的Plg+/+小鼠和Plg-/-小鼠各5只，各组小鼠体重基本相同。小鼠在实验过程中都饲以同样的食物和水。取股骨于4％多聚甲醛固定24小时，进行Micro CT扫描，测定骨密度。
结果显示，在12-30周龄期间随着周龄增大Plg+/+小鼠股骨密度逐渐增加，而Plg-/-小鼠股骨皮质骨密度(10A)、松质骨密度(图10B)、小梁骨密度 (图10C)以及总骨密度(图10D)逐渐减小。在此期间，Plg+/+小鼠股骨密度高于Plg-/-小鼠，在20周龄时两个品系小鼠骨密度差异显著，并且随着周龄增大，二者差异越来越显著。说明纤溶酶原参与股骨矿物质代谢调节，并在一定时期发挥重要作用。
实施例11纤溶酶原缺乏小鼠股骨矿物含量减少
取12-13、20-21、29-30周龄的Plg+/+小鼠和Plg-/-小鼠各5只，各组小鼠体重基本相同。小鼠在实验过程中都饲以同样的食物和水。取股骨于4％多聚甲醛固定24小时，进行Micro CT扫描，测定骨矿物含量。
结果显示，在12-30周龄期间随着周龄增大Plg+/+小鼠股骨不同部分矿物含量变化不大或逐步增加，而Plg-/-小鼠股骨松质骨(11B)和小梁骨矿物含量逐渐减小。在此期间，Plg+/+小鼠股骨矿物含量高于Plg-/-小鼠，在20周龄时两个品系小鼠股骨皮质骨(11A)、小梁骨(11C)和总骨(11D)骨矿物含量差异显著，并且随着周龄增大，二者骨矿物含量差异越来越显著。说明纤溶酶原参与股骨矿物质代谢调节，并在一定时期发挥重要作用。
实施例12纤溶酶原缺乏小鼠腰椎骨密度降低
取12-13、20-21、29-30周龄的Plg+/+小鼠和Plg-/-小鼠各5只，各组小鼠体重基本相同。小鼠在实验过程中都饲以同样的食物和水。取腰椎骨骨于4％多聚甲醛固定24小时，进行Micro CT扫描，测定骨密度。
结果显示，在12-30周龄期间随着周龄增大Plg+/+小鼠腰椎骨骨密度逐渐增大，而Plg-/-小鼠腰椎骨皮质骨密度(12A)、松质骨密度(图12B)、小梁骨密度(图12C)以及总骨密度(图12D)逐渐减小。在此期间，Plg+/+小鼠骨密度高于Plg-/-小鼠，在12周龄时两个品系小鼠腰椎骨骨密度差异显著，并且随着周龄的增大，二者差异越来越显著。说明纤溶酶原参与腰椎骨骨密度调节，并在一定时期发挥重要作用。
实施例13纤溶酶原缺乏小鼠腰椎骨矿物含量减少
取12-13、20-21、29-30周龄的Plg+/+小鼠和Plg-/-小鼠各5只，各组小鼠体重基本相同。小鼠在实验过程中都饲以同样的食物和水。取腰椎骨于4％多聚甲醛固定24小时，进行Micro CT扫描，测定骨矿物含量。
结果显示，在12-30周龄期间随着周龄增大Plg+/+小鼠腰椎骨矿物含量变化不大，而Plg-/-小鼠腰椎骨皮质骨矿物含量(13A)、松质骨矿物含量(图 13B)、小梁骨矿物含量(图13C)以及总骨矿物含量(图13D)逐渐减小。在此期间，Plg+/+小鼠骨矿物含量高于Plg-/-小鼠，在20周龄时两个品系小鼠腰椎骨松质骨和皮质骨区域矿物含量差异显著，并且随着周龄增大，二者差异越来越显著。说明纤溶酶原参与腰椎骨骨矿物质代谢调节，并在一定时期发挥重要作用。
实施例14纤溶酶原缺乏对小鼠血清碱性磷酸酶活性的影响
取12-13、20-21、29-30周龄的Plg+/+小鼠和Plg-/-小鼠各5只，各组小鼠体重基本相同。小鼠在实验过程中都饲以同样的食物和水。所有小鼠摘除眼球取血，离心取上清。用碱性磷酸酶检测试剂盒检测血清中碱性磷酸酶活性。
结果显示，Plg+/+小鼠血清碱性磷酸酶活性在12-30周龄时虽有波动但变化并不显著，而Plg-/-小鼠血清碱性磷酸酶的活性随着周龄的增大逐渐的降低；Plg+/+小鼠血清碱性磷酸酶的活性明显高于Plg-/-小鼠，两个品系小鼠血清碱性磷酸酶活性在12周龄时就出现明显的差异，并且随着周龄的增大，差异越来越显著(图14)。该结果提示纤溶酶原可能促进成骨细胞的活性，促进骨重建。
实施例15纤溶酶原降低动脉粥样硬化ApoE小鼠血钙浓度
6周龄ApoE雄性小鼠13只饲喂高脂高胆固醇饲料16周，诱导动脉粥样硬化[39,40]。在给药前三天每只小鼠取血50μL，检测总胆固醇浓度，并据其将小鼠随机分为两组，给溶媒PBS对照组7只以及给纤溶酶原组6只。开始给药定为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药期间小鼠继续饲喂高脂饲料。在第30天小鼠禁食16小时，第31天摘除眼球取血，离心获得上清，用以检测血清钙的浓度。血钙检测采用钙检测试剂盒(南京建成生物工程研究所，货号C004-2)并按照说明书方法进行检测。
结果显示，给纤溶酶原组小鼠血钙浓度要明显的低于给溶媒PBS对照组，且统计差异显著(图15)。说明纤溶酶原能够降低ApoE动脉粥样硬化模型小鼠血钙的含量。
实施例16纤溶酶原改善动脉粥样硬化ApoE小鼠主动脉窦钙化
6周龄ApoE雄性小鼠13只饲喂高脂高胆固醇饲料(南通特洛菲，TP2031)16周，诱导动脉粥样硬化[39,40]。在给药前三天每只小鼠取血50μL，检测总胆固醇浓度，并据其将小鼠随机分为两组，给溶媒PBS对照组7只以及给纤溶酶原组6只。开始给药定为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药期间小鼠继续饲喂高脂饲料。于给药的第31天处死小鼠，取材心脏于4％多聚甲醛固定24-48小时，15％、30％蔗糖脱水沉底，OCT包埋，冰冻切片厚度8μm，茜素红S染色3分钟。切片在40倍光学显微镜下观察。
茜素红染色能够显示钙化的程度。结果显示，给纤溶酶原组(图16B)小鼠主动脉窦钙沉积明显少于给溶媒PBS对照组(图16A)。说明纤溶酶原能够改善动脉粥样硬化中主动脉窦钙沉积。
实施例17纤溶酶原对AopE动脉粥样硬化模型小鼠股骨骨密度的影响
6周龄AopE雄性小鼠19只饲喂高脂高胆固醇饲料(南通特洛菲，TP2031)16周，诱导动脉粥样硬化[39,40]。在给药前三天每只小鼠取血50μL，检测总胆固醇浓度，并据其将小鼠随机分为两组，给溶媒PBS对照组9只以及给纤溶酶原组10只。开始给药定为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药期间小鼠继续饲喂高脂饲料。于给药的第11天每组各取5只小鼠，处死后取材股骨于4％多聚甲醛固定。给药的31天处死剩余小鼠，取材股骨于4％多聚甲醛固定。取材的股骨进行Micro CT扫描，测定骨密度。
动脉粥样硬化与骨质疏松相关性早已有报道，高血脂是动脉粥样硬化的重要致病因素。近来研究表明，载脂蛋白E(apolipoprotein E，ApoE)不仅影响脂代谢，而且与骨密度、骨丢失以及骨质疏松性骨折有关[41,42]。
结果显示，给药10天后给纤溶酶原组小鼠股骨密度明显高于给溶媒PBS对照组，且松质骨密度(图17B)和总骨密度(图17D)差异显著(*表示P<0.05)；给药30天后给纤溶酶原组小鼠松质骨密度、小梁骨密度(图17C)和总骨密度相较于给溶媒PBS对照组显著升高，且统计差异显著(*表示P<0.05)。皮质骨密度(图17A)两组无明显差异。说明纤溶酶原能够促进动脉粥样硬化模型小鼠骨密度升高，改善动脉粥样硬化伴随的骨质疏松。
已有研究表明，血管钙化的本质是血管平滑肌细胞向成骨细胞表型的转化以及血管组织向骨组织的转化。而血管钙化的形成也和骨矿物质丢失具有显著的相关性[10]。综合上述实施例16和17的实验结果可以看出，纤溶酶原能够在降低动脉壁钙沉积的同时增强骨密度。对骨质疏松症及心血管疾病的防治具有重要意义。
实施例18纤溶酶原对AopE动脉粥样硬化模型小鼠膝关节组织结构的保护作用
6周龄AopE雄性小鼠7只饲喂高脂高胆固醇饲料(南通特洛菲，TP2031)16周，诱导动脉粥样硬化[39,40]。在给药前三天每只小鼠取血50μL，检测总胆固醇浓度，并据其将小鼠随机分为两组，给溶媒PBS对照组3只以及给纤溶酶原组4只。开始给药定为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药期间小鼠继续饲喂高脂饲料。于给药的31天处死小鼠，取材股骨于4％多聚甲醛固定。然后用酸性脱钙液(用超纯水配制体积分数为8％盐酸和10％甲酸的脱钙液)脱钙3.5小时。然后石蜡包埋，8μm切片行H&E染色，切片在100倍(A、D)、200倍(B、C、E、F)光学显微镜下观察。
结果显示，给溶媒PBS对照组(图18A-C)软骨表面轻度纤维化(细箭头标识)，骨小梁(三角标识)明显变细，粗细不均匀，软骨组织(星形标识)排列紊乱，生长板(粗箭头标识)层次紊乱，软骨细胞轻度减少，潮线基本清晰；给纤溶酶原组(图18D-F)关节软骨表面基本正常，潮线清晰，骨小梁粗细均匀，生长板结构清晰，层次规则而可分。说明纤溶酶原能够改善ApoE动脉粥样硬化模型小鼠膝关节的状况。
实施例19纤溶酶原对卵巢切除-地塞米松骨质疏松模型小鼠体重的影响
8-10周龄C57雌性小鼠17只称量体重，小鼠根据体重随机分为两组，正常对照组3只和模型组14只。模型组小鼠按照50mg/kg体重腹腔注射戊巴比妥钠麻醉，小鼠脱去背部两侧毛发以70％的酒精以及碘酊消毒，切开皮肤、背部肌肉和腹膜，以小镊子轻轻将白色发亮脂肪团拉出切口外，分离脂肪团，便可见到卵巢。先将卵巢下端输卵管用丝线结扎，然后摘除卵巢。切口缝合后，外部敷以消炎粉。同法摘除另一侧卵巢。正常对照小鼠仅在相同位置切开，不做卵巢切除。卵巢切除14天后，模型组小鼠根据体重随机 分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各7只。模型组小鼠按照125μg/只腹腔注射地塞米松，5天/周注射频率，共注射12天诱导骨质疏松[43]，正常对照组小鼠不做注射处理。注射地塞米松的同时小鼠开始给药，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药16天，正常对照组小鼠不注射纤溶酶原或PBS。开始给药定为第1天，第17天测量小鼠体重。
结果显示，给溶媒PBS对照组小鼠体重明显轻于正常对照组，而给纤溶酶原组体重明显高于给溶媒PBS对照组，且统计差异显著(P<0.05)(图19)。说明纤溶酶原能够显著促进卵巢切除及注射地塞米松诱导骨质疏松模型小鼠体重恢复。
实施例20纤溶酶原对卵巢切除-地塞米松骨质疏松模型小鼠股骨的影响
8-10周龄C57雌性小鼠14只称量体重。所有小鼠按照50mg/kg体重腹腔注射戊巴比妥钠麻醉，小鼠脱去背部两侧毛发以70％的酒精以及碘酊消毒，切开皮肤、背部肌肉和腹膜，以小镊子轻轻将白色发亮脂肪团拉出切口外，分离脂肪团，便可见到卵巢。先将卵巢下端输卵管用丝线结扎，然后摘除卵巢。切口缝合后，外部敷以消炎粉。同法摘除另一侧卵巢。卵巢切除14天后，小鼠根据体重随机分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各7只。两组小鼠按照125μg/只腹腔注射地塞米松，5天/周注射频率，共注射12天诱导骨质疏松[43]。造模同时小鼠开始给药，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药16天。开始给药定为第1天，第17天处死取材股骨于4％多聚甲醛固定液中固定。进行Micro CT扫描，测定股骨各项指标。
结果显示，给纤溶酶原组小鼠股骨的松质骨、小梁骨以及总骨体积(图20A)和骨矿物含量(图20B)均大于给溶媒PBS对照组，且均统计差异显著(*表示P<0.05)。说明纤溶酶原能够促进骨质疏松模型小鼠股骨的松质骨、小梁骨和总骨体积的增加及矿物质的沉积，改善骨质疏松。
实施例21纤溶酶原改善卵巢切除-地塞米松骨质疏松模型小鼠股骨结构
8-10周龄C57雌性小鼠17只称量体重，小鼠根据体重随机分为两组，正常对照组3只和模型组14只。模型组小鼠按照50mg/kg体重腹腔注射戊巴比 妥钠麻醉，小鼠脱去背部两侧毛发以70％的酒精以及碘酊消毒，切开皮肤、背部肌肉和腹膜，以小镊子轻轻将白色发亮脂肪团拉出切口外，分离脂肪团，便可见到卵巢。先将卵巢下端输卵管用丝线结扎，然后摘除卵巢。切口缝合后，外部敷以消炎粉。同法摘除另一侧卵巢。正常对照小鼠仅在相同位置切开，不做卵巢切除。卵巢切除14天后，模型组小鼠按照125μg/只腹腔注射地塞米松，5天/周注射频率，共注射12天诱导骨质疏松[43]，正常对照组小鼠不做注射处理。地塞米松注射完成后，模型组小鼠根据体重随机分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各7只。模型建立后(地塞米松注射完成第二天)，小鼠开始给药。给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药16天，正常对照组小鼠不注射纤溶酶原或PBS。开始给药定为第1天，第17天处死取材股骨于4％多聚甲醛固定液中固定。取材的股骨进行Micro CT扫描，测定股骨密度。
骨密度
结果显示，给溶媒PBS对照组小鼠股骨皮质骨、松质骨、小梁骨和总骨骨密度均小于正常对照组；而给纤溶酶原组小鼠各部分骨密度大于给溶媒PBS对照组。趋势明确，但由于小鼠数量少，造成统计差异只是接近显著。可预期增加小鼠数量会出现统计学差异(图21A)。
骨矿物含量
结果显示，给溶媒PBS对照组小鼠股骨各部分骨矿物含量均小于正常对照组；而给纤溶酶原组小鼠各部分骨矿物含量均大于给溶媒PBS对照组。趋势明确，但由于小鼠数量少，造成统计差异只是接近显著。可预期增加小鼠数量会出现统计学差异(图21B)。
骨体积
结果显示，给溶媒PBS对照组小鼠股骨的小梁骨体积小于正常对照组；而给纤溶酶原组小鼠股骨小梁骨体积大于给溶媒PBS对照组。趋势明确，但由于小鼠数量少，造成统计差异只是接近显著。可预期增加小鼠数量会出现统计学差异(图21C)。
综上所述，纤溶酶原能够明显改善骨质疏松，促进股骨各部分骨密度和骨量的增加，并且对小梁骨改善作用尤为明显。
实施例22纤溶酶原改善卵巢切除-地塞米松骨质疏松模型小鼠膝关节组织结构的状况
8-10周龄C57雌性小鼠14只称量体重。所有小鼠按照50mg/kg体重腹腔注射戊巴比妥钠麻醉，小鼠脱去背部两侧毛发以70％的酒精以及碘酊消毒，切开皮肤、背部肌肉和腹膜，以小镊子轻轻将白色发亮脂肪团拉出切口外，分离脂肪团，便可见到卵巢。先将卵巢下端输卵管用丝线结扎，然后摘除卵巢。切口缝合后，外部敷以消炎粉。同法摘除另一侧卵巢。卵巢切除14天后，小鼠根据体重随机分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各7只。两组小鼠按照125μg/只腹腔注射地塞米松，5天/周注射频率，共注射12天诱导骨质疏松[43]。造模同时小鼠开始给药，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药16天。开始给药定为第1天，第17天处死取材膝关节于4％多聚甲醛固定液中固定。然后用酸性脱钙液(用超纯水配制体积分数为8％盐酸和10％甲酸的脱钙液)脱钙3.5小时。然后石蜡包埋，3μm切片行H&E(A、B)和Safrain O染色(C、D)，切片在100倍光学显微镜下观察。
结果显示，给溶媒PBS组(图22A、C)小鼠膝关节骨小梁(箭头标识)明显变细，断裂，出现较大面积的无骨小梁骨髓腔，髓腔增大，骨小梁的连接中断，生长板下骨细胞轻度减少(三角形标识)；给纤溶酶原组(图22B、D)骨小梁部分变细，较之于PBS组，骨小梁连续性较好，较粗，没有较大面积的无骨小梁区域，软骨组织层次结构也较规则。说明纤溶酶原能明显改善骨质疏松模型小鼠膝关节组织结构的状况。
实施例23纤溶酶原增加卵巢切除-地塞米松骨质疏松模型小鼠膝关节成骨细胞活性
8-10周龄C57雌性小鼠14只称量体重。所有小鼠按照50mg/kg体重腹腔注射戊巴比妥钠麻醉，小鼠脱去背部两侧毛发以70％的酒精以及碘酊消毒，切开皮肤、背部肌肉和腹膜，以小镊子轻轻将白色发亮脂肪团拉出切口外，分离脂肪团，便可见到卵巢。先将卵巢下端输卵管用丝线结扎，然后摘除卵巢。切口缝合后，外部敷以消炎粉。同法摘除另一侧卵巢。卵巢切除14天后，小鼠根据体重随机分为两组，给纤溶酶原组和给溶媒PBS对照 组，每组各7只。两组小鼠按照125μg/只腹腔注射地塞米松，5天/周注射频率，共注射12天诱导骨质疏松[43]。造模同时小鼠开始给药，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药16天。开始给药定为第1天，第17天处死小鼠取材膝关节于固定液中固定。固定液配方:2％多聚甲醛，0.075mol/L赖氨酸，0.01mol/L过碘酸钠。固定后4℃PBS洗液梯度洗涤各12小时，然后置于4℃脱钙液中脱钙2周，每5天换一次脱钙液。脱钙完成后4℃PBS洗液梯度洗涤12小时，膝关节经酒精梯度脱水和二甲苯透明后进行石蜡包埋。切片3um，脱蜡复水，氯化镁缓冲液4℃孵育过夜。碱性磷酸酶底物溶液室温孵育1小时，苏木素复染2分钟。流水冲洗5分钟，60℃烤30分钟，中性树胶封片，切片在200倍光学显微镜下观察。
结果显示，给溶媒PBS对照组小鼠(图23A、C)膝关节软骨组织(细箭头标识)和生长板(粗箭头标识)碱性磷酸酶着色明显少于给纤溶酶原组(图23B、D)。说明纤溶酶原促进骨质疏松模型小鼠膝关节成骨细胞活性增加。
实施例24纤溶酶原降低卵巢切除骨质疏松模型小鼠血钙浓度
8-10周龄Plg+/+雌性小鼠11只。小鼠按照50mg/kg体重腹腔注射戊巴比妥钠麻醉，小鼠脱去背部两侧毛发以70％的酒精以及碘酊消毒，切开皮肤、背部肌肉和腹膜，以小镊子轻轻将白色发亮脂肪团拉出切口外，分离脂肪团，便可见到卵巢。先将卵巢下端输卵管用丝线结扎，然后摘除卵巢。切口缝合后，外部敷以消炎粉。同法摘除另一侧卵巢[44,45]。卵巢切除65天后，小鼠称重，并根据体重随机分为两组，给纤溶酶原组6只和给溶媒PBS对照组5只，并开始给药。给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药11天。开始给药定为第1天，第12天摘除眼球取血，离心取上清，检测血钙浓度。血钙检测采用钙检测试剂盒(南京建成生物工程研究所，货号C004-2)并按照说明书方法进行检测。
结果显示，给纤溶酶原组小鼠血清钙浓度明显低于给溶媒PBS对照组，且统计差异显著(*表示P<0.05)(图24)。说明纤溶酶原能够明显地降低卵巢切除骨质疏松模型小鼠血钙的浓度。
实施例25纤溶酶原升高卵巢切除骨质疏松模型小鼠血磷浓度
8-10周龄Plg+/+雌性小鼠11只。小鼠按照50mg/kg体重腹腔注射戊巴比妥钠麻醉，小鼠脱去背部两侧毛发以70％的酒精以及碘酊消毒，切开皮肤、背部肌肉和腹膜，以小镊子轻轻将白色发亮脂肪团拉出切口外，分离脂肪团，便可见到卵巢。先将卵巢下端输卵管用丝线结扎，然后摘除卵巢。切口缝合后，外部敷以消炎粉。同法摘除另一侧卵巢[44,45]。卵巢切除65天后，小鼠称重，并根据体重随机分为两组，给纤溶酶原组6只和给溶媒PBS对照组5只，并开始给药。给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药11天。开始给药定为第1天，第12天摘除眼球取血，离心取上清，检测血磷浓度。血磷检测采用磷检测试剂盒(南京建成生物工程研究所，货号C006-3)并按照说明书方法进行检测。
结果显示，给纤溶酶原组小鼠血清磷浓度明显高于给溶媒PBS对照组，且统计差异显著(*表示P<0.05)(图25)。说明纤溶酶原能够明显的升高卵巢切除骨质疏松模型小鼠血磷的浓度。
实施例26纤溶酶原增加3％胆固醇高脂血症模型小鼠膝关节成骨细胞活性
9周龄雄性C57小鼠16只饲喂3％胆固醇高脂饲料(南通特洛菲)4周，诱导高脂血症[46,47]，此模型定为3％胆固醇高脂血症模型。成模后的小鼠继续饲喂3％胆固醇高脂饲料。在给药前三天每只小鼠取血50μL，检测总胆固醇，并根据总胆固醇浓度和体重随机分为两组，每组各8只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药20天。在第20天小鼠禁食16小时，第21天处死小鼠取材膝关节于固定液中固定。固定液配方:2％多聚甲醛，0.075mol/L赖氨酸，0.01mol/L过碘酸钠。固定后4℃PBS洗液梯度洗涤各12小时，然后置于4℃脱钙液中脱钙2周，每5天换一次脱钙液。脱钙完成后4℃PBS洗液梯度洗涤12小时，膝关节经酒精梯度脱水和二甲苯透明后进行石蜡包埋。切片3um，脱蜡复水，氯化镁缓冲液4℃孵育过夜。碱性磷酸酶底物溶液室温孵育1小时，苏木素复染2分钟。流水冲洗5分钟，60℃烤30分钟，中性树胶封片，切片在200倍光学显微镜下观察。
高脂血症是一种脂代谢紊乱，并能引起一系列并发症的疾病。近年来多项研究发现高脂血脂是骨质疏松和动脉粥样硬化的共同病因[48,49]。
结果显示，给纤溶酶原组(图26B、D)小鼠膝关节碱性磷酸酶着色(箭头标识)明显多于给溶媒PBS对照组(图26A、C)，且统计差异显著(图26E)。说明纤溶酶原增加3％胆固醇高脂血症模型小鼠膝关节成骨细胞活性。
实施例27纤溶酶原改善卵巢切除-地塞米松骨质疏松模型小鼠膝关节组织结构状况
8-10周龄C57雌性小鼠14只称量体重。小鼠按照50mg/kg体重腹腔注射戊巴比妥钠麻醉，小鼠脱去背部两侧毛发以70％的酒精以及碘酊消毒，切开皮肤、背部肌肉和腹膜，以小镊子轻轻将白色发亮脂肪团拉出切口外，分离脂肪团，便可见到卵巢。先将卵巢下端输卵管用丝线结扎，然后摘除卵巢。切口缝合后，外部敷以消炎粉。同法摘除另一侧卵巢。卵巢切除14天后，模型组小鼠按照125μg/只腹腔注射地塞米松，5天/周注射频率，共注射12天诱导骨质疏松[43]。地塞米松注射完成后，小鼠根据体重随机分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各7只。模型建立后(地塞米松注射完成第二天)，小鼠开始给药。给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药16天。开始给药定为第1天，第17天处死取材膝关节于4％多聚甲醛固定液中固定。然后用酸性脱钙液(用超纯水配制体积分数为8％盐酸和10％甲酸的脱钙液)脱钙3.5小时。然后石蜡包埋，3μm切片行H&E(A、B)和Safrain O染色(C、D)，切片在100倍光学显微镜下观察。
结果显示，给溶媒PBS对照组(图27A、C)小鼠骨小梁(箭头标识)明显变细，断裂，出现较大面积的无骨小梁骨髓腔，骨小梁的连接中断，关节表面部分纤维化，生长板下成骨区成骨组织明显减少(三角形标识)；给纤溶酶原组(图27B、D)骨小梁部分变细，较之于PBS对照组，骨小梁连续性较好，没有较严重的断裂，没有较大面积的无骨小梁区域，软骨组织层次结构也较规则，潮线清晰。说明给予纤溶酶原能明显改善骨质疏松模型小鼠膝关节的组织结构状况。
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Claims (35)

  1. 一种预防和治疗骨质疏松及其相关病症的方法，包括给药受试者治疗有效量的纤溶酶原。
  2. 权利要求1的方法，其中所述骨质疏松包括原发性骨质疏松和继发性骨质疏松。
  3. 权利要求1的方法，其中所述原发性骨质疏松包括绝经后骨质疏松和老年性骨质疏松。
  4. 权利要求1或2的方法，其中所述继发性骨质疏松包括继发于内分泌疾病、风湿性疾病、胃肠道疾病，以及药物治疗引起的骨质疏松。
  5. 权利要求4的方法，其中所述继发性骨质疏松包括糖皮质激素、原发性甲状旁腺功能亢进、甲状腺功能亢进、原发性胆汁性肝硬化、性腺功能减低、糖尿病、高血压、动脉粥样硬化、慢性肾脏疾病、类风湿关节炎、系统性红斑狼疮、强直性脊柱炎、骨关节炎、性腺激素治疗、抗癫痫药治疗、化疗药物治疗引起的骨质疏松。
  6. 一种预防和治疗疾病并发的骨质疏松的方法，包括给药受试者有效量的纤溶酶原，其中所述疾病并发的骨质疏松包括糖皮质激素、原发性甲状旁腺功能亢进、甲状腺功能亢进、原发性胆汁性肝硬化、性腺功能减低、糖尿病、高血压、动脉粥样硬化、慢性肾脏疾病、类风湿关节炎、系统性红斑狼疮、强直性脊柱炎、骨关节炎、性腺激素治疗、抗癫痫药治疗、化疗药物治疗并发的骨质疏松。。
  7. 一种预防骨质疏松骨折的方法，包括给药易患骨质疏松的受试者、处于患骨质疏松高风险的受试者或诊断患有骨质疏松的受试者有效量的纤溶酶原预防骨折的发生。
  8. 权利要求7的方法，其中所述受试者包括患有糖皮质激素、原发性甲状旁腺功能亢进、甲状腺功能亢进、原发性胆汁性肝硬化、性腺功能减低、糖尿病、高血压、动脉粥样硬化、慢性肾脏疾病、类风湿关节炎、系统性红斑狼疮、强直性脊柱炎或骨关节炎的受试者。
  9. 权利要求7的方法，其中所述受试者包括正接受性腺激素治疗、抗癫痫药治疗或化疗药物治疗的受试者。
  10. 一种增强成骨细胞活性的方法，包括给药受试者有效量的纤溶酶原。
  11. 一种调控骨矿物质代谢的方法，包括给药受试者有效量的纤溶酶原。
  12. 权利要求11的方法，所述调控包括降低血钙水平、升高血磷水平，促进钙在骨基质中的沉积和/或降低钙在血管壁、内脏的沉积。
  13. 权利要求1-12任一项的方法，其中所述纤溶酶原与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。
  14. 权利要求1-12任一项的方法，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。
  15. 权利要求1-12任一项的方法，所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。
  16. 权利要求1-12任一项的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。
  17. 权利要求1-12任一项的方法，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。
  18. 权利要求1-12任一项的方法，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。
  19. 权利要求13-18任一项的方法，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。
  20. 权利要求1-19任一项的方法，其中所述纤溶酶原是人天然纤溶酶原。
  21. 权利要求1-20任一项的方法，其中所述受试者是人。
  22. 权利要求1-21任一项的方法，其中所述受试者缺乏或缺失纤溶酶原。
  23. 权利要求22的方法，其中所述缺乏或缺失是先天的、继发的和/或局部的。
  24. 一种用于权利要求1-23任一项的方法的纤溶酶原。
  25. 一种药物组合物，其包含药学上可接受的载剂和用于权利要求1-23中任一项所述方法的纤溶酶原。
  26. 一种预防性或治疗性试剂盒，其包含：(i)用于权利要求1-23中任一项所述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)。
  27. 根据权利要求26所述的试剂盒，其中所述构件为注射器或小瓶。
  28. 权利要求26或27的试剂盒，其还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施权利要求1-23中任一项所述方法。
  29. 一种制品，其包含：
    含有标签的容器；和
    包含(i)用于权利要求1-23中任一项所述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施权利要求1-23中任一项所述方法。
  30. 权利要求26-28中任一项的试剂盒或权利要求29的制品，还包含另外的一个或多个构件或容器，该构件或容器中含有其它药物。
  31. 权利要求30的试剂盒或制品，其中所述其它药物包括治疗骨质疏松的其它药物或治疗与骨质疏松并发的其它疾病的药物。
  32. 包含纤溶酶原的用于治疗骨质疏松的药剂。
  33. 包含纤溶酶原的用于治疗骨质疏松的药物组合物，试剂盒、制品。
  34. 纤溶酶原用于治疗骨质疏松的用途。
  35. 本发明还涉及纤溶酶原在制备上述权利要求1-23任一项的方法中使用的药物、药物组合物、制品、试剂盒中的用途。
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