CN106890323A

CN106890323A - A kind of method for preventing and treating liver tissue injury and its associated conditions

Info

Publication number: CN106890323A
Application number: CN201611170627.7A
Authority: CN
Inventors: 李季男
Original assignee: Shenzhen Life Science Research Institute Co Ltd
Current assignee: Shenzhen Life Science Research Institute Co Ltd
Priority date: 2015-12-18
Filing date: 2016-12-16
Publication date: 2017-06-27

Abstract

Purposes the present invention relates to plasminogen in terms for the treatment of and/or eliminating hepatic injury, a new therapy approach is provided to treat different types of hepatic injury.

Description

A kind of method for preventing and treating liver tissue injury and its associated conditions

Technical field

The present invention relates to plasminogen or fibrinolysin in terms of liver tissue injury caused by prevention and/or treatment a variety of causes Purposes, and then be treatment liver tissue injury and its associated conditions brand-new therapeutic strategy is provided.

Background

Hepatic injury or liver tissue injury are liver parenchyma lesions caused by a variety of causes, are thin liver tissues inflammatory, liver A series of general name of pathological changes such as born of the same parents' denaturation, necrosis, liver tissue fibrosis., there are inflammation, liver extravasated blood, virus common the reason for Infection, poisoning, medicine, radiation etc..Some diseases also occur together the damage of liver organization cell, such as diabetes, hepatitis, blood high Pressure, atherosclerosis etc..

The reason for medicine is very common cause hepatic tissue to damage one.The common drug bag for causing liver organization to damage Include：Antituberculotic：Rimactazid, ethambutol etc.；Antineoplastic：It is phonetic that endoxan, first ammonia fold purine, 5- fluorine urine Pyridine, carboplatin, cis-platinum etc.；Downgrade blood-lipoids：Statins (Atorvastatin, Lovastatin), fenofibrate, Clofibrate, nicotinic acid Deng；Steroid hormone：Estrogen drugs, oral contraceptive, male anabolic hormone etc.；Cardiovascular drugs：Amiodarone, Hua Fa Make, calcium ion antagonist etc.；Antirheumatic：Indocin, fenbufen, aspirin, Indomethacin etc.；Antibiotic：Chloramphenicol, ROX, ketoconazole, PCs, sulfamido etc.；Claritin：Fenazil (phenergan), chlorphenamine (chlorpheniramine), Loratadine (Clarityne) etc.；Anti-ulcer medicament：Cimetidine, ranitidine, famotidine etc.；Antimycotic medicine is for example sharp Ba Weilin etc..

Alcohol is grave danger of liver, and long-term or discontinuity heavy drinking can cause liver tissue injury, and drinking amount is big, hold Continuous Ganderwa is more long, and its consequence is more serious.Alcohol can directly poison liver cell, influence its structure and function.

Alcoholic liver injury is a kind of slow poisoning hepatic injury, is due to liver diseases caused by long-term heavy drinking. Initial stage is usually expressed as fatty liver, and then can develop into alcoholic hepatitis, liver fibrosis and cirrhosis.Its main clinical characteristics is Nausea and vomiting, jaundice, can there are liver enlargement and tenderness.Extensive necrosis of liver cells can be induced during serious excessive drinking, or even liver function declines Exhaust.AML is one of common liver diseases of China, serious harm people's health^[1]。

In addition to toxic liver injury caused by alcohol, other " hepatotropic poisons ", such as chemical toxic substances in environment And some drugses can also cause hepatic injury.As the important removing toxic substances organ of human body, liver, with arteria hepatica and vena hepatica double blood Liquid is supplied.Chemical substance can be converted by intestines and stomach portal vein or body circulation into liver, therefore liver is easily changed Learn the toxicant infringement in thing.There are some in the Nature and human industry's production process to the virose material of liver, Referred to as " hepatotropic poison ", these poisonous substances are universal susceptible in crowd, and incubation period is short, and the process of lesion is direct with the amount of chemical substance Correlation, can cause the different degrees of necrosis of liver cells of liver, steatosis, cirrhosis.Pathological manifestations include (1) steatosis. Carbon tetrachloride, yellow phosphorus etc. may interfere with the synthesis and transhipment of lipoprotein, form fatty liver.(2) peroxidatic reaction of lipid, this is poisoning Property hepatic injury special representing form, such as carbon tetrachloride is metabolized the intermediate product for producing a kind of oxidability very strong, leads in vivo The lipid peroxidation on biomembrane is caused, the phosphatide of film is destroyed, changes the structure and function of cell.(3) cholestasia reaction, mainly It is impaired with liver plasma membrane and microvillus, cause bile acid excretion obstacle relevant^[2]。

Radiation can also cause liver organization to damage.In general, radiation source is natural or the high energy of artificial energy source's generation Electromagnetic wave or high energy particle.Irradiation may all cause tissue damage for a long time for heavy dose of ray moment irradiation or low dosage ray, Chromosome, the enzyme of the energy damages cell of radiation, make the normal function of cell get muddled.

Diabetic keratopathy liver tissue injury refers to the lesion of the liver histological and changes of function caused by diabetes.Known sugars The hepatic injury that urine disease can cause includes：Liver enzyme is abnormal, and it can cause carbon dioxide accumulation in liver cell, acid poisoning, oxygen for subtracting Less, oxygen consumption increases, and increases liver transaminases activity, and Disorders of bilirubin metabolism, severe one can cause necrosis of liver cells；Fat Liver, in all causes of disease for causing fatty liver, diabetes account for the 3rd, wherein 21%~78% diabetic is with fat Liver；The illness rate of virus hepatitis is about 2~4 times of normal person in hepatitis, hardening and liver cancer, wherein diabetic, primary The incidence of property liver cancer is about 4 times of normal person.Diabetic hepatopathy not only damages the quality of life of millions of patients, Nursing needed for also resulting in a huge burden cost and medical health system intensity simultaneously.

The virus infection of liver is also to cause a common cause of hepatic injury, for example virus B hepatitis, the third type disease Malicious hepatitis, penta type viral hepatitis etc..

Sludging can also cause liver organization to damage in liver.Blood deposits in liver and is mainly drawn by following several factors Rise：Veno-occlusive disease of the liver VOD, the Ji Yali syndromes of Ahmedabad one, chronic right heart insufficiency and constrictive pericarditis.

Any disease for causing the Inferior Vena Cava Blood time heart to be obstructed can all cause liver extravasated blood, such as rheumatic heart valve disease, Chronic constrictive pericarditis, hypertensive cardiopathy, ischemic heart disease, pulmonary heart disease, congenital heart disease etc..

Centrilobular area, centrilobular venous congestion, expansion, sinus hepaticus degrees of expansion and liver are initially involved in congestive hepatic injury Sinus is far and near and different away from centrilobular vein, and centrilobular liver cell is pressurized, and deforms and atrophy, is in particle in cytoplasm Sample becomes, and has karyopycnosis, and nuclear fission, meronecrosis is calm with brown pigmentation, and brown pigmentation is located at centrilobular, may be because becoming silted up Caused by courage, the liver parenchyma degeneration necrosis most serious of adjacent central vein, with the exacerbation of extravasated blood, slough extends to door area, sternly There is more normal hepatic tissue in weight extravasated blood patient Jin Men areas, and with time lengthening, the reticular fibre around central vein can be collapsed, can See that reticular fibre tissue and fine fibre beam extend to another central vein from central vein.

For the treatment main control and treatment including the cause of disease at present of liver tissue injury, and supportive treatment.It is long-term with Come, scientists always search for having the medicine of direct, good repairing effect to damaging hepatic tissue.The present invention is also carried out to this In-depth study.Experiment discovery, naturally occurring a kind of protein material in human body, plasminogen is to poisoning, radiation, chemotherapy Liver tissue injury has good repair caused by medicine and diabetes.Plasminogen be expected to turn into treatment liver tissue injury and A kind of new strategy of its associated conditions.

Plasminogen (plasminogen, plg) is the nonactive precursor of fibrinolysin, is a kind of single chain glycoprotein, by 791 Individual amino acid composition, molecular weight is about 92kDa^[3,^4].Plasminogen is main in liver synthesis, is largely present in extracellular fluid.Blood Content of plasminogen is about 2 μM in slurry.Therefore plasminogen is one of its proteolytic activity in tissue and body fluid huge Potential source^[5,^6].There are two kinds of molecular forms in plasminogen：Glutamic acid-plasminogen (Glu-plasminogen) and bad ammonia Acid-plasminogen (Lys-plasminogen).The plasminogen of natural secretion and uncracked form has an amino terminal (N- ends) glutamic acid, therefore it is referred to as glutamic acid-plasminogen.However, in the presence of fibrinolysin, glutamic acid-plasminogen Hydrolysis turns into lysine-plasminogen at Lys76-Lys77.Compared with glutamic acid-plasminogen, lysine-plasminogen There is affinity higher with fibrin, it is possible to which speed higher is activated by PA.The plasminogen of both forms Arg560-Val561 peptide bonds can be cut by uPA or tPA, cause the formation of the dichain proteins enzyme fibrinolysin of disulfide bond^[7].It is fine The former amino terminus portion of lyase includes five homologous three rings, i.e., so-called kringle, carboxy-terminal sections include protease knot Structure domain.Some kringle contain the bad ammonia that mediation plasminogen interacts with fibrin and its inhibitor α 2-AP specificity Sour binding site.Latest find one is the plasminogen fragment of 38kDa, is blood vessel including kringle1-4 Effective inhibitor of generation.This fragment is named as angiostatin, can be produced by several protease hydrolytic plasminogens.

Invention summary

On the one hand, prevention and/or treatment subject's liver tissue injury are being prepared the present invention relates to plasminogen or fibrinolysin And its purposes in medicine, product, the medicine box of associated conditions.The invention further relates to a kind of pharmaceutical methods, including by plasminogen It is prepared into medicine, the system of prevention and/or treatment subject's liver tissue injury and its associated conditions jointly with pharmaceutical acceptable carrier Product, medicine box.

In one embodiment, the liver tissue injury and its associated conditions are that the liver that radiation or chemical substance cause damages Injure its associated conditions.In one embodiment, the hepatic injury and its associated conditions that radiation or chemical substance cause are cancer The used chemicotherapy method for the treatment of and medicine.In one embodiment, the radiation is due to accident or building ring Radiation caused by other events such as border.In one embodiment, the liver tissue injury and its associated conditions are Poisoning liver Damage and its associated conditions.In one embodiment, the toxic liver injury is including " hepatotropic poison " including alcohol The toxic liver injury for causing.In one embodiment, the liver tissue injury and its associated conditions cause for diabetes, It is one of complication of diabetes.In one embodiment, the liver tissue injury and its associated conditions are virus infection liver Caused by dirty caused by hepatitis, such as hepatitis A virus, hepatitis type B virus, HCV, Hepatitis D virus, penta type Hepatitis caused by hepatitis viruse.In one embodiment, the liver tissue injury and its associated conditions are drug induced hepatic injury And its associated conditions.In one embodiment, the liver tissue injury and its associated conditions are that (liver becomes silted up sludging in liver Blood) caused by.In one embodiment, the liver tissue injury and its associated conditions are diabetic keratopathy hepatic injury and its correlation Illness, toxic liver injury and its associated conditions, drug induced hepatic injury or its associated conditions, radiativity hepatic injury and its related diseases Disease, viral infection hepatic injury and its associated conditions, congestive hepatic injury and its associated conditions.In one embodiment, institute State liver tissue injury and its associated conditions include caused by liver tissue injury dysfunction of liver, liver enzyme extremely, uncomfortable liver area and Tenderness, hepatomegaly, splenomegaly, hepatosplenomegaly, hepatitis, fatty liver, cholangitis, cirrhosis, hepatonecrosis and liver cancer.

In an embodiment, the plasminogen and sequence 2,6,8,10 or 12 have at least 80%, 85%, 90%th, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is plasminogen activity. One embodiment, the plasminogen is comprising activities of endothelial tissue plasminogen fragment and still with plasmin The protein of zymogen activity.

In an embodiment, the plasminogen be selected from Glu- plasminogens, Lys- plasminogens, Small plasminogen, fibrillin lyase original, δ-plasminogen or its any combination.It is described in an embodiment Plasminogen or fibrinolysin applied systemically or topically, including surface, intravenous, intramuscular, subcutaneous, suction, intraspinal tube, local note Penetrate, intra-articular injection or rectal administration.In an embodiment, the associated conditions of the diabetic keratopathy hepatic injury or Poisoning liver The associated conditions of damage include：Liver enzyme exception, uncomfortable liver area and tenderness, hepatomegaly, splenomegaly, hepatosplenomegaly, hepatitis, fat Liver, cholangitis, cirrhosis, hepatonecrosis and liver cancer.In an embodiment, the diabetic keratopathy hepatic injury and its associated conditions are The big blood vessel that is caused by diabetes, thin vessels, microangiopathies cause.In an embodiment, the plasminogen can be with one Plant or various other medicines are co-administered.In an embodiment, the other medicines include：Hepatic, antidiabetic Thing, antithrombotic reagent, anticoagulant, blood lipid-lowering medicine, medicament against cardiovascular disease, anti-infectives.

In an embodiment, the subject is mammal, is preferably people.

In one embodiment, the hepatic injury caused by diabetes is big blood vessel, the small blood caused by diabetes Pipe, microangiopathies cause.

In one embodiment, subject's fibrinolysin or plasminogen are low.Specifically, it is described low Under be inborn, secondary and/or part.

In one embodiment, plasminogen and sequence 2,6,8,10 or 12 have at least 80%, 85%, 90%th, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is plasminogen activity. In one embodiment, plasminogen is addition, deletion and/or substitution 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- 4th, 1-3,1-2,1 amino acid, and still there is the protein of plasminogen activity.In one embodiment, Plasminogen is comprising activities of endothelial tissue plasminogen fragment and still with the protein of plasminogen activity.At one In embodiment, plasminogen is selected from Glu- plasminogens, Lys- plasminogens, small fibrinolysin Former, fibrillin lyase original, δ-plasminogen or its any combination.In one embodiment, plasminogen is selected from such as Under conservative substitution variant：Glu- plasminogens, Lys- plasminogens, small plasminogen, δ-fibre Lyase is former or fibrillin lyase is former.In one embodiment, plasminogen behaviour natural fiber plasmin Original, such as plasminogen shown in sequence 2 it is straight to homologue, for example, the fiber from primate or rodent Plasmin is former straight to homologue, such as, from gorilla, rhesus macaque, mouse, ox, horse, the plasminogen of dog is straight to same It is thing.Most preferably, the amino acid sequence of plasminogen of the invention is as shown in sequence 2,6,8,10 or 12.

In one embodiment, the plasminogen is applied with appropriate peptide carrier or combination of stabilizers.At one In embodiment, the plasminogen with daily 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 (being calculated with per kilogram of body weight) or 0.0001- 2000mg/cm²、0.001-800mg/cm²、0.01-600mg/cm²、0.1-400mg/cm²、1-200mg/cm²、1-100mg/ cm²、10-100mg/cm²The dosage of (being calculated with body surface area every square centimeter) is applied, and is preferably at least repeated once, preferably at least Apply daily.In the case of local application, above-mentioned dosage according to circumstances can also be adjusted further.

In one embodiment, the plasminogen is preferably applied by administered either systemically or locally by following approach： Surface, intravenous, intramuscular, subcutaneous, suction, intraspinal tube, local injection, intra-articular injection or by rectum.In an embodiment party In case, the local administration is carried out by liver region using the conduit containing plasminogen.

On the one hand, the present invention relates to a kind of method prevented and/or treat subject's liver tissue injury and its associated conditions, Plasminogen or fibrinolysin including subject's effective dose is administered.It is used to prevent the invention further relates to plasminogen or fibrinolysin And/or the purposes for the treatment of subject's liver tissue injury and its associated conditions.

In one embodiment, the liver tissue injury and its associated conditions are that the liver that radiation or chemical substance cause damages Injure its associated conditions.In one embodiment, the hepatic injury and its associated conditions that radiation or chemical substance cause are cancer The used chemicotherapy method for the treatment of and medicine.In one embodiment, the radiation is due to spoke caused by accident Penetrate.In one embodiment, the liver tissue injury and its associated conditions are toxic liver injury and its associated conditions.One In individual embodiment, the toxic liver injury is the toxic liver injury caused including " hepatotropic poison " including alcohol. In one embodiment, the liver tissue injury and its associated conditions cause for diabetes, are one of complication of diabetes. In one embodiment, caused by the liver tissue injury and its associated conditions are for hepatitis caused by virus infected liver, for example Hepatitis caused by hepatitis A virus, hepatitis type B virus, HCV, Hepatitis D virus, HEV. In one embodiment, the liver tissue injury and its associated conditions are drug induced hepatic injury and its associated conditions.In a reality Apply in scheme, the liver tissue injury and its associated conditions are caused sludgings in liver (liver extravasated blood).In an implementation In scheme, the liver tissue injury and its associated conditions be diabetic keratopathy hepatic injury and its associated conditions, toxic liver injury and Its associated conditions, drug induced hepatic injury or its associated conditions, radiativity hepatic injury and its associated conditions, viral infection hepatic injury And its associated conditions, congestive hepatic injury and its associated conditions.In one embodiment, the liver tissue injury and its correlation Illness includes dysfunction of liver, liver enzyme exception, uncomfortable liver area and tenderness, hepatomegaly, splenomegaly, liver caused by liver tissue injury Splenomegaly, hepatitis, fatty liver, cholangitis, cirrhosis, hepatonecrosis and liver cancer.

In an embodiment, the plasminogen and sequence 2,6,8,10 or 12 have at least 80%, 85%, 90%th, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is plasminogen activity. One embodiment, the plasminogen is comprising activities of endothelial tissue plasminogen fragment and still with plasmin The protein of zymogen activity.In an embodiment, the plasminogen is selected from Glu- plasminogens, Lys- fiber eggs White lyase is former, small plasminogen, fibrillin lyase are former, δ-plasminogen or its any combination.In an implementation Scheme, the plasminogen or fibrinolysin applied systemically or topically, including surface, intravenous, intramuscular, it is subcutaneous, suction, canalis spinalis Interior, local injection, intra-articular injection or rectal administration.In an embodiment, the associated conditions of the diabetic keratopathy hepatic injury Or the associated conditions of toxic liver injury include：Liver enzyme is abnormal, uncomfortable liver area and tenderness, hepatomegaly, splenomegaly, liver spleen swell Greatly, hepatitis, fatty liver, cholangitis, cirrhosis, hepatonecrosis and liver cancer.In an embodiment, the diabetic keratopathy hepatic injury and Its associated conditions is that the big blood vessel that is caused by diabetes, thin vessels, microangiopathies cause.In an embodiment, the fibre Lyase original can be co-administered with one or more other medicines.In an embodiment, the other medicines include：Hepatoprotective agent Thing, antidiabetic medicine, antithrombotic reagent, anticoagulant, blood lipid-lowering medicine, medicament against cardiovascular disease, anti-infectives.

In an embodiment, the subject is mammal, is preferably people.

In one embodiment, plasminogen and sequence 2,6,8,10 or 12 have at least 80%, 85%, 90%th, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is plasminogen activity. In one embodiment, plasminogen is addition, deletion and/or substitution 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- 4th, 1-3,1-2,1 amino acid, and still there is the protein of plasminogen activity.In one embodiment, Plasminogen is comprising activities of endothelial tissue plasminogen fragment and still with the protein of plasminogen activity.At one In embodiment, plasminogen is selected from Glu- plasminogens, Lys- plasminogens, small fibrinolysin Former, fibrillin lyase original, δ-plasminogen or its any combination.In one embodiment, plasminogen is selected from such as Under conservative substitution variant：Glu- plasminogens, Lys- plasminogens, small plasminogen, δ-fibre Lyase is former or fibrillin lyase is former.In one embodiment, plasminogen behaviour natural fiber plasmin Original, such as plasminogen shown in sequence 2 it is straight to homologue, for example, the fiber from primate or rodent Plasmin is former straight to homologue, such as, from gorilla, rhesus macaque, mouse, ox, horse, the plasminogen of dog are straight to same It is thing.Most preferably, the amino acid sequence of plasminogen of the invention is as shown in sequence 2,6,8,10 or 12.

On the one hand, the present invention relates to a kind of fibrinolytic for preventing and/or treating subject's liver tissue injury and its associated conditions Proenzyme or fibrinolysin, the pharmaceutical composition comprising the plasminogen or fibrinolysin or comprising the plasminogen or fibrinolysin Product or medicine box.

In an embodiment, the subject is mammal, is preferably people.

In one embodiment, the plasminogen or fibrinolysin are sub-divided in container.It is preferred that, the product or medicine box Other medicines are also included, is divided in other containers of the medicine box.The medicine box can also include operation instructions, illustrate the fibre Lyase original can be used for treating liver tissue injury and its associated conditions, specifically, the Diabetic liver that such as diabetes cause is damaged And its liver damages caused by associated conditions, toxic liver injury and its associated conditions, drug induced hepatic injury or its associated conditions, radiation Injure its associated conditions, virus and infect hepatic injury and its associated conditions caused by the hepatic injury and its associated conditions, extravasated blood for causing, And can further illustrate, the plasminogen or fibrinolysin can before other medicines or therapy are applied, meanwhile, and/ Or apply afterwards.

The present invention clearly covers all combinations for belonging to the technical characteristic between embodiment of the present invention, and these groups Technical scheme after conjunction is clearly disclosed in this application, just as above-mentioned technical proposal is individually and clearly disclosing. In addition, the present invention also clearly covers all sub-combinations of each embodiment and its key element, and it is disclosed herein, just as every One such sub-combination is independent and clearly disclosed herein the same.

Detailed description of the invention

" diabetic keratopathy hepatic injury " refers to the lesion of the liver histological and changes of function caused by diabetes.Its it is main by Big blood vessel that diabetes cause, thin vessels, microangiopathies cause.The hepatic injury that known diabetes can cause includes：Liver enzyme Abnormal, it can cause carbon dioxide accumulation in liver cell, acid poisoning, oxygen for reducing, oxygen consumption increases, and make liver transaminases activity Increase, Disorders of bilirubin metabolism, severe one can cause necrosis of liver cells；Fatty liver, in all causes of disease for causing fatty liver, glycosuria Disease accounts for the 3rd, wherein 21%~78% diabetic is with fatty liver；Hepatitis, hardening and liver cancer, wherein diabetic The illness rate of middle virus hepatitis is about 2~4 times of normal person, and the incidence of primary carcinoma of liver is about 4 times of normal person.

" chemical damage " or " toxic liver injury " refers to the hepatic lesion as caused by chemically Hepatoxic substance.This A little chemical substances include chemical toxic substances and some drugses in alcohol, environment.In the Nature and human industry's production process There are some to the virose material of liver, referred to as " hepatotropic poison ", these poisonous substances universal susceptible, incubation period in crowd Short, the process of lesion is directly related with the amount of chemical substance, can cause the different degrees of necrosis of liver cells of liver, steatosis, Cirrhosis.

" hepatotropic poison " refers to the general name to the virose material of liver.Alcohol is most common " close liver poison in life Thing ".In addition to alcohol, chemical toxic substances and some drugses in environment can also cause hepatic injury.As the important removing toxic substances of human body Organ, liver has arteria hepatica and vena hepatica double blood supply.Chemical substance can be entered by intestines and stomach portal vein or body circulation Enter liver to be converted, therefore liver is easily subject to the toxicant infringement in chemicals.The Nature and human industry produced There are some to the virose material of liver, referred to as " hepatotropic poison " in Cheng Zhongjun.They enter liver, can cause liver difference journey The necrosis of liver cells of degree, steatosis, cirrhosis.Pathological manifestations include (1) steatosis.Carbon tetrachloride, yellow phosphorus etc. may interfere with The synthesis of lipoprotein and transhipment, form fatty liver.(2) peroxidatic reaction of lipid, this is the Special Manifestations shape of toxic liver injury Formula, such as carbon tetrachloride are metabolized the intermediate product for producing a kind of oxidability very strong in vivo, cause the lipid peroxy on biomembrane Change, destroy the phosphatide of film, change the structure and function of cell.(3) cholestasia reaction, mainly receives with liver plasma membrane and microvillus Damage, cause bile acid excretion obstacle relevant.

During " drug induced hepatic injury " is medicine use, because of medicine in itself and/or its metabolite or due to special body Hepar damnification caused by supersensitivity or the tolerance reduction of confrontation medicine is referred to as drug induced hepatic injury, also known as Drug liver Disease, clinically can behave as various acute and chronic hepatitis, and the lighter can voluntarily recover after being discontinued, and severe one possibility threat to life, need are positive Treatment, rescue." drug induced hepatic injury " can occur in healthy person in the past without hepatitis history or just have serious disease originally With patient；Can occur in medication excess, in the case of may also occur at normal usage.

" radiativity hepatic injury " refers to that high-energy ionization radiation causes including α, β particle, gamma-rays, χ rays and neutron ray A kind of radioactive damage.Irradiation may all cause tissue damage for a long time for heavy dose of ray moment irradiation or low dosage ray, Chromosome, the enzyme of the energy damages cell of radiation, make the normal function of cell get muddled.

" viral infection hepatic injury " is hepatic injury general name caused by virus infection.The virus infection is common A type liver Infection caused by scorching virus, hepatitis type B virus, HCV, Hepatitis D virus, HEV.

Liver organization damages lesion caused by " congestive hepatic injury " deposits for blood in liver.It is any to cause inferior caval vein The disease that the blood time heart is obstructed can all cause liver extravasated blood, such as rheumatic heart valve disease, chronic constrictive pericarditis, hypertensive cerebral Heart disease, ischemic heart disease, pulmonary heart disease, congenital heart disease etc..

" plasminogen " is the zymogen forms of fibrinolysin, according to the sequence in swiss prot, by the day containing signal peptide Right people source plasminogen amino acid sequence (sequence 4) calculates and is made up of 810 amino acid, and molecular weight is about 92kD, mainly in liver Dirty middle synthesis and the glycoprotein that can circulate in blood, encode the cDNA sequence of the amino acid sequence as shown in sequence 3.Total length Plasminogen include seven domains：The Pan Apple (PAp) of serine protease domain, N-terminal positioned at C-terminal Domain and 5 Kringle domains (Kringle1-5).With reference to the sequence in swiss prot, its signal peptide includes residual Base Met1-Gly19, PAp include that residue Glu20-Val98, Kringle1 include residue Cys103-Cys181, Kringle2 bag Including residue Glu184-Cys262, Kringle3 includes that residue Cys275-Cys352, Kringle4 include residue Cys377- Cys454, Kringle5 include residue Cys481-Cys560.According to NCBI data, serine protease domain includes residue Val581-Arg804。

Glu- plasminogens are the plasminogens of Native full-length, are made up of 791 amino acid and (do not contain 19 amino acid Signal peptide), the cDNA sequence of the sequence is encoded as shown in sequence 1, its amino acid sequence is as shown in sequence 2.In vivo, also exist A kind of is that the Lys- plasminogens so as to be formed are hydrolyzed from the 76-77 amino acids of Glu- plasminogens, such as the institute of sequence 6 Show, encode the cDNA sequence of the amino acid sequence as shown in sequence 5.Δ-plasminogen (δ-plasminogen) is total length fibrinolytic Proenzyme has lacked the fragment of Kringle2-Kringle5 structures, only contains Kringle1 and serine protease domain^[8,9], there is text Offer the amino acid sequence (sequence 8) for reporting δ-plasminogen^[9], encode the cDNA sequence such as sequence 7 of the amino acid sequence.It is small Plasminogen (Mini-plasminogen) is made up of Kringle5 and serine protease domain, and it includes residue document report Val443-Asn791 (being initial amino acid with the Glu residues for not containing the Glu- plasminogen sequences of signal peptide)^[10], its ammonia Base acid sequence encodes the cDNA sequence of the amino acid sequence as shown in sequence 9 as shown in sequence 10.And Microplasminogen (Micro-plasminogen) only contain serine protease domain, there is its amino acid sequence of document report to include residue Ala543-Asn791 (being initial amino acid with the Glu residues for not containing the Glu- plasminogen sequences of signal peptide)^[11], also have Patent document CN102154253A reports that its sequence includes residue Lys531-Asn791 (not contain the Glu- fibrinolytics of signal peptide The Glu residues of proenzyme sequence are initial amino acid), this patent sequence reference patent document CN102154253A, its amino acid sequence Row encode the cDNA sequence of the amino acid sequence as shown in sequence 11 as shown in sequence 12.

" fibrinolysin " of the invention is used interchangeably with " fibrinolysin ", " fibrinoclase ", and implication is identical； " plasminogen " is used interchangeably with " plasminogen ", " plasminogen ", and implication is identical.

It will be understood by those skilled in the art that all technical schemes of plasminogen of the present invention are applied to fibrinolysin, therefore, The technical scheme of present invention description covers plasminogen and fibrinolysin.

In cyclic process, plasminogen uses the nonactive conformation of closing, but when thrombus or cell surface is bound to, Under the mediation of plasminogen activator (plasminogen activator, PA), it is changed into the activity in open conformation Fibrinolysin.Fibrin clot further can be hydrolyzed to fibrin degradation product (FDP) and D- dimerization by active fibrinolysin Body, and then thrombus.Wherein the PAp domains of plasminogen include the weight for maintaining plasminogen to be in nonactive closing conformation Determinant is wanted, and KR domains can then be combined with the lysine residue being present on acceptor and substrate.It is known various to make It is the enzyme of plasminogen activator, including：Tissue plasminogen activator (tPA), uPA (uPA), Kallikrein and Hageman factor (Hageman factor (HF)) etc..

" activities of endothelial tissue plasminogen fragment " refers to that in plasminogen protein, can be combined and played with the target sequence in substrate The active fragment of proteolysis function.Technical scheme the present invention relates to plasminogen was covered with activities of endothelial tissue plasminogen fragment generation For the technical scheme of plasminogen.Activities of endothelial tissue plasminogen fragment of the present invention is the serine protease comprising plasminogen The protein in domain, it is preferable that activities of endothelial tissue plasminogen fragment of the present invention has at least 80% comprising sequence 14 and sequence 14, 90%th, the protein of the amino acid sequence of 95%, 96%, 97%, 98%, 99% homology.Therefore, fibrinolytic of the present invention Proenzyme includes containing the activities of endothelial tissue plasminogen fragment and remains in that the albumen of the activities of endothelial tissue plasminogen.

At present, include for plasminogen in blood and its activity determination method：To tissue plasminogen Activator activity detection (t-PAA), the detection (t-PAAg) of plasma tissue PLA antigen, to blood Starch detection (plgA), the detection (plgAg) of plasma tissue plasminogen antigen, the plasma tissue fiber of tissue plasminogen activity The detection of Plasminogen Activators Inhibitor Activity, the inspection of plasma tissue Plasminogen activator inhibitor antigen Survey, plasma fibrin lyase-antiplasmin complex detects (PAP).The detection method of most common of which is color development Substrate method：Add streptokinase (SK) and chromophoric substrate to by inspection blood plasma, by the PLG in inspection blood plasma in the presence of SK, be transformed into PLM, the latter acts on chromophoric substrate, then with spectrophotometric determination, absorbance increase with plasminogen activity into Direct ratio.In addition also can be using immuno-chemical method, gel electrophoresis, immunoturbidimetry, radioimmunodiffusion etc. to the fibre in blood The molten zymogen activity of fibrillarin is measured.

" ortholog thing or lineal homologue (ortholog) " refers to the homologue between different plant species, both same including albumen Source thing also includes DNA homology thing, also referred to as straight homologues, Paralog thing.It is referred specifically in different plant species by same ancestors Gene evolution and come albumen or gene.Plasminogen of the invention includes the natural plasminogen of people, also including from not Plasminogen ortholog thing infraspecific, with activities of endothelial tissue plasminogen or lineal homologue.

" conservative substitution variant " refer to one of them given amino acid residue change but do not change protein or enzyme it is whole Body conformation and function, this is included but is not limited to the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor parent of similar characteristic (such as acid, alkalescence, hydrophobicity, etc.) Amino acid in Amino Acids in Proteins sequence.Amino acid with similarity is well-known.For example, arginine, group Propylhomoserin and lysine are hydrophilic basic amino acids and can exchange.Equally, isoleucine is hydrophobic amino acid, then can be bright Propylhomoserin, methionine or valine are replaced.Therefore, two albumen of identity function or the similitude of amino acid sequence may not Together.For example, based on MEGALIGN algorithms 70% to 99% similarity (homogeneity)." conservative substitution variant " also includes passing through BLAST or fasta algorithm determine the polypeptide or enzyme of the amino acid identities with more than 60%, if can be more preferable up to more than 75%, Preferably up to more than 85%, or even it is optimal up to more than 90%, and has compared with natural or parent protein or enzyme identical Or similar property or function substantially.

" separation " plasminogen refers to the plasminogen protein for separating and/or reclaiming from its natural surroundings.In some realities Apply in scheme, the plasminogen can purify (1) extremely more than the 90%, purity (by weight) more than 95% or more than 98%, As by determined by Lowry methods, such as, more than 99% (by weight), (2) are to being enough to by using rotating cup sequence analysis Instrument obtains at least 15 degree of residue of N-terminal or internal amino acid sequence, or (3), to homogeney, the homogeney is by making With the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Coomassie blue or silver staining under reproducibility or non-reducing conditions (SDS-PAGE) determine.The plasminogen of separation also includes being prepared from recombinant cell by biotechnology, and by extremely The plasminogen that a few purification step is separate.

Term " polypeptide ", " peptide " and " protein " is used interchangeably herein, and refers to the polymerization of the amino acid of any length It is form, its amino acid that can include genetic coding and non-genetic coding, chemistry or biochemical modification or derivatization Amino acid, and the polypeptide with modified peptide backbone.The term includes fusion protein, including but not limited to heterologous amino The fusion protein of acid sequence, with heterologous and homologous leader sequences (with and without N-terminal methionine residues) fusions；Deng Deng.

It is defined as introducing breach when necessary on " amino acid sequence identity percentage (%) " with reference to polypeptide sequence After realizing largest percentage sequence identity, and when any conservative replacement not being considered as into a part for sequence identity, candidate With the percentage with reference to the amino acid residue identical amino acid residue in polypeptide sequence in sequence.To determine percent amino acid The contrast of sequence identity purpose can be realized with the various ways in the range of art technology, such as using publicly available Computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can be certainly Surely it is used for the suitable parameter of aligned sequences, including any algorithm that maximum contrast needs is realized to institute's comparative sequences total length.However, For the purposes of the present invention, amino acid sequence identity percent value is to compare computer program ALIGN-2 using sequence to produce 's.

In the case of using ALIGN-2 come comparing amino acid sequence, amino acid sequence A is given relative to given amino acid The % amino acid sequence identities of sequence B (or can be expressed as having or comprising relative to or for given amino acid sequence Arrange the given amino acid sequence A of a certain % amino acid sequence identities of B) it is calculated as below：

Fraction X/Y multiplies 100

Wherein X is that scoring is the amino of identical match in the A and B of the program are compared by alignment programs ALIGN-2 The number of sour residue, and wherein Y is the sum of the amino acid residue in B.It will be appreciated that in the length and ammonia of amino acid sequence A In the case of the length of base acid sequence B is unequal, % amino acid sequence identities of the A relative to B can be not equal to B relative to A % amino acid sequence identities.Unless expressly stated otherwise, all % amino acid sequence identities values used herein are all It is, according to described in the preceding paragraph, to be obtained using ALIGN-2 computer programs.

As used in this article, term " treatment " and " treatment " refers to the desired pharmacology of acquisition and/or physiologic effect.The effect Fruit can be prevention disease or its symptom wholly or in part, and/or partially or completely cure diseases and/or its symptom, and wrap Include：A () prevention disease occurs in subject, the subject can have the procatarxis of disease, but not yet be diagnosed as tool There is disease；B () suppresses disease, that is, block its formation；(c) mitigates disease and/or its symptom, that is, cause disease and/or its disease Shape disappears.

Term " individuality ", " subject " and " patient " is used interchangeably herein, and refers to mammal, including but not limited to Mouse (rat, mouse), non-human primates, people, dog, cat, ungulate (such as horse, ox, sheep, pig, goat) etc..

" therapeutically effective amount " or " effective dose " refers to and is enough to when applying mammal or other subjects to treat disease Realize the amount to the prevention of disease and/or the plasminogen for the treatment of." therapeutically effective amount " can be according to the fibrinolysin for being used The former, disease of subject to be treated and/or the order of severity of its symptom and age, body weight etc. and change.

The preparation of plasminogen of the present invention

Plasminogen can be separated and purified for further treatment purposes from nature, it is also possible to by the change of standard Peptide symthesis technology is learned to synthesize.When by chemically synthesized polypeptide, can be synthesized through liquid phase or solid phase.Solid phase peptide synthssis (SPPS) (the C-terminal amino acid of sequence is wherein attached into insoluble holder, then remaining amino in sequential addition sequence Acid) it is the method for being adapted to plasminogen chemical synthesis.Various forms of SPPS, such as Fmoc and Boc can be used to synthesize fibrinolysin It is former.Technology for synthesis in solid state is described in Barany and Solid-Phase Peptide Synthesis；The 3-284 pages in The Peptides:Analysis, Synthesis, Biology. volume 2：Special Methods in Peptide Synthesis, Part A., Merrifield, wait J.Am.Chem.Soc., 85:2149-2156(1963)；Stewart etc., Solid Phase Peptide Synthesis,2nd ed.Pierce Chem.Co.,Rockford,Ill.(1984)；With Ganesan A.2006Mini Rev.Med Chem.6:The 2005Protein such as 3-10 and Camarero JA Pept Lett.12:In 723-8.In short, processing small insoluble porous bead with the functional element for being built with peptide chain thereon.In idol After connection/de-protected repetitive cycling, the free N-terminal amine of solid phase that will be attached is coupled with the single Amino Acid Unit protected by N.So Afterwards, this element is deprotected, exposes the new N-terminal amine that can be attached with other amino acid.Peptide is remained fixed in solid phase, it Cut away afterwards.

Plasminogen of the invention can be produced using Standard recombinant methods.For example, by the nucleic acid of encoding plasminogen In insertion expression vector, it is set to be operatively connected with the regulating and controlling sequence in expression vector.Expression regulation sequence is included but is not limited to Promoter (such as naturally associated or heterologous promoter), signal sequence, enhancer element and transcription terminator.Expression Regulation and control can be eukaryotic promoter system in carrier, the carrier can convert or transfect eukaryotic host cell (such as COS or Chinese hamster ovary celI).Once during carrier mixed into suitable host, it is being suitable for the high level expression and plasminogen of nucleotide sequence Collection and purifying under conditions of maintain host.

Suitable expression vector is generally in host organisms as episome or the integration portion as host chromosome DNA Divide and replicate.Generally, expression vector contain selection marker thing (for example amicillin resistance, hygromycin resistance, tetracyclin resistance, Kalamycin resistance or neomycin resistance) contributing to those cells converted with desired DNA sequence dna to external source to detect.

Escherichia coli (Escherichia coli) can be used for cloning the protokaryon place of theme antibody coding polynucleotides The example of chief cell.Other microbial hosts being adapted for use with include bacillus, such as bacillus subtilis (Bacillus Subtilis) and other enterobacteriaceaes (Enterobacteriaceae), such as Salmonella (Salmonella), sand Lei Shi Pseudomonas (Serratia) and various pseudomonas (Pseudomonas) species.In these prokaryotic hosts, it is also possible to generate Expression vector, it would generally contain the expression control sequenc (such as replication orgin) compatible with host cell.In addition, can exist being permitted Many known promoters, such as lactose promoter system, tryptophan (trp) promoter systems, beta-lactamase promoter systems, Or the promoter systems from phageλ.Promoter would generally control table reach, optionally in the case of operator sequence, and And with ribosome bind site sequence etc., to start and complete transcription and translation.

Other microorganisms, such as yeast can also be used for expression.Yeast (such as saccharomyces cerevisiae (S.cerevisiae)) and finish Red yeast (Pichia) is the example of suitable yeast host cell, wherein suitable carrier has expression control sequence as needed Row (such as promoter), replication orgin, terminator sequence etc..Typical promoter includes glycerol 3-phosphate acid kinase and other sugars Solution enzyme.Induction type yeast starts in particularly including from alcohol dehydrogenase, different cell pigment C and responsible maltose and galactolipin profit The promoter of enzyme.

Outside microorganism, mammalian cell (mammalian cell for example cultivated in cell culture in vitro) also may be used For expressing and generate plasminogen of the invention (polynucleotides of the anti-Tau antibody of such as encoding schemes).Referring to Winnacker,From Genes to Clones,VCH Publishers,N.Y.,N.Y.(1987).Suitable mammal Host cell includes Chinese hamster ovary celI system, various Cos cell lines, HeLa cells, myeloma cell line and inverted B cell or miscellaneous Hand over knurl.Expression vector for these cells can include expression control sequenc, such as replication orgin, promoter and enhancer (Queen etc., Immunol.Rev.89:49 (1986)), and required machining information site, such as ribosome bind site, RNA splice sites, polyadenylation site, and transcription terminator sequences.The example of suitable expression control sequenc is white exempting from The derivative promoter such as epidemic disease globulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus.Referring to Co etc., J.Immunol.148:1149(1992)。

Once synthesis (chemistry or recombination form), can be affine according to the standard schedule of this area, including ammonium sulfate precipitation Post, column chromatography, high performance liquid chroma- tography (HPLC), gel electrophoresis etc. purify plasminogen of the present invention.The plasminogen It is substantially pure, for example, at least about 80% to 85% is pure, at least about 85% to 90% is pure, at least about 90% to 95% is pure , or 98% to 99% is pure or purer, such as without pollutant, the pollutant such as cell fragment, except theme antibody with Outer macromolecular, etc..

Pharmaceutical formulation

Can by will have needed for purity plasminogen and optional pharmaceutical carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences, 16 editions, Osol, A.ed. (1980)) be mixed to form lyophilized formulations or The aqueous solution prepares treatment preparaton.Acceptable carrier, excipient, stabilizer are nontoxic to receptor under dosage used and concentration Property, and including buffer such as phosphate, citrate and other organic acids；Antioxidant includes ascorbic acid and methionine； Preservative (such as stearyl dimethyl benzyl ammonium chloride；Hexamethonium chloride；Benzalkonium chloride (benzalkonium Chloride), benzethonium chloride；Phenol, butanol or phenmethylol；Alkyl parabens such as methyl or propyl p-hydroxybenzoate Ester；Catechol；Resorcinol；Cyclohexanol；3- amylalcohols；Metacresol)；Low molecular weight polypeptide (less than about 10 residues)；Albumen Matter such as seralbumin, gelatin or immunoglobulin；Hydrophilic polymer such as polyvinylpyrrolidone；Amino acid such as glycine, paddy Glutamine, asparagine, histidine, arginine or lysine；Monose, disaccharides and other carbohydrate include glucose, sweet Dew sugar or dextrin；Chelating agent such as EDTA；Carbohydrate such as sucrose, mannitol, fucose or sorbierite；Into salt counter ion such as sodium；Metal Compound (such as zinc-albumen composition)；And/or nonionic surfactant, such as TWEENTM, PLURONICSTM or poly- second Glycol (PEG).It is preferred that lyophilized anti-VEGF antibodies preparaton is described in WO 97/04801, it is included in herein as ginseng Examine.

Preparaton of the invention can also contain the specific illness that need to treat needed for more than one reactive compound, preferably Complementary activities and be free from side effects each other those.For example, antihypertensive medicine, antiarrhythmic medicine, controlling Treat medicine of diabetes etc..

Plasminogen of the invention can be wrapped in the microcapsules prepared by such as condensation technique or interfacial polymerization, example Such as, colloidal drug delivery systems (for example, liposome, albumin microsphere, microemulsion, nano particle and Nano capsule) can be embedded in In or the hydroxymethyl cellulose inserted in macro emulsion or gelatin-microcapsule and poly- (methyl methacrylate) microcapsules in. These technologies are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)。

Plasminogen of the invention for vivo medicine-feeding is necessarily aseptic.This can be by freeze-drying and again Realized easily by degerming membrane filtration before or after preparation.

Plasminogen of the invention can prepare sustained release preparation.The appropriate example of sustained release preparation includes having definite shape and contain There are the penetrating matrix of solid hydrophobic polymers half of glycoprotein, such as film or microcapsules.Sustained-release matrix example includes polyester, hydrogel (such as poly- (2- hydroxyethyl-methacrylates) (Langer, J.Biomed.Mater.Res., 15:167-277(1981)； Langer,Chem.Tech.,12:98-105 (1982)) or poly- (vinyl alcohol), polyactide (United States Patent (USP) 3773919, EP 58, 481), the copolymer (Sidman, etc. Biopolymers 22 of Pidolidone and γ ethyl-L-glutamates:547 (1983)), no Degradable ethylene vinyl acetate (ethylene-vinyl acetate) (Langer, etc. ibid), or it is degradable Poly lactic coglycolic acid such as Lupron DepotTM (by poly lactic coglycolic acid and leucyl proline (leuprolide) microsphere of the injectable of acetic acid esters composition), and poly- D- (-) -3- hydroxybutyric acids.Polymer such as ethene-second Vinyl acetate and lactic-co-glycolic acid energy sustained release molecule more than 100 days, and the time of some hydrogels release proteins compared with It is short.Can be designed according to Related Mechanism makes protein stabilized rational strategy.For example, if it find that the mechanism of cohesion is by sulphur Intermolecular S -- S is formed for Disulfide interchange, then can by modify sulfhydryl residue, from acid solution freeze, control humidity, Stabilization is realized using suitable additive and the specific polymer matrix composition of exploitation.

Administration and dosage

Can by different modes, for example by intravenous, intraperitoneal, subcutaneous, encephalic, intrathecal, intra-arterial (for example via Arteria carotis), intramuscular, intranasal, surface or intradermal administration or spinal cord or brain deliver to realize the administration of pharmaceutical composition of the present invention.Gas Aqueous or other solution and preservative and isotonic agent of purifying of the sol preparation such as nose spray preparation comprising activating agent.By such system Agent is adjusted to the pH and isotonic state compatible with schneiderian membrane.

Prepared product for parenteral administration includes sterile aqueous or non-aqueous solution, suspension and emulsion.It is non-aqueous molten The example of agent is propane diols, polyethylene glycol, vegetable oil such as olive oil, and injectable organic ester, such as ethyl oleate.Aqueous carrier bag Include water, alcohol/aqueous solution, emulsion or suspension, including salt solution and buffer medium.Parenteral medium is molten comprising sodium chloride Liquid, woods grignard dextrose, dextrose and sodium chloride or fixing oil.Intravenous vehicles include liquid and nutritional supplement, electrolysis Matter supplement, etc..Can also there are preservative and other additives, such as, antimicrobial, antioxidant, chelating Agent and inert gas, etc..

Medical worker can determine dosage based on various clinical factors.It is such as known in medical domain, any patient's Dosage depend on many factors, including patient build, body surface area, age, the particular compound to be applied, sex, administration Number of times and path, general health and the other medicines being administered simultaneously.The dosage of pharmaceutical composition of the present invention comprising plasminogen Scope can be, for example, daily about 0.0001 to 2000mg/kg, or about 0.001 to 500mg/kg (such as 0.02mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 10mg/kg, 50mg/kg etc.) subject's body weight.For example, dosage can be 1mg/kg body weight or 50mg/kg body weight or the scope in 1-50mg/kg, or at least 1mg/kg.Higher or lower than this exemplary model Including the dosage for enclosing is also covered by, above-mentioned factor is especially considering that.Middle dosage in above range is also contained in the present invention In the range of.Subject can apply this daily, every other day, weekly or according to any other schedule determined by empirical analysis Class dosage.Exemplary dosage schedule includes continuous several days 1-10mg/kg.Reality is needed during medicament administration of the invention When assessment, the therapeutic effect and security of periodical evaluation diabetes hepatopathy and its associated conditions.

Treatment effect and Therapeutic safety

After one embodiment of the invention is directed to use with plasminogen treatment subject, to treatment effect and treatment safety The judgement of property.Determination methods wherein to the treatment effect are included but is not limited to：1) to the inspection of subject's liver function, for example To zymetology level in patient's body for example serum AST (ALT), glutamic-pyruvic transaminase (AST), total bilirubin, directly Whether the levels such as bilirubin, indirect bilirubin, albumin, globulin, cholinesterase, alkaline phosphatase, transpeptidase are in normal Value scope is checked, after being treated to subject using the plasminogen in the present invention, it is contemplated that above-mentioned liver function index will Recover to normal value or improved, for example, glutamic-pyruvic transaminase (ALT)：0~40 μ/L, glutamic-oxalacetic transaminease (AST)：0~40 μ/ L, glutamyl transferase (GGT)：Less than 40 units, total bilirubin：3.4~20.5 μm of ol/L；2) when to subject's factor Between (PT) and mobility (PTA) checked：PT is the important indicator for reflecting liver clotting factor complex functionality, and PTA is that PT is surveyed The conventional method for expressing of definite value, to judging that liver disease progression and prognosis have a larger value, wherein PTA progressive is down to less than 40% and is One of important diagnostic standard of hepatic failure,<20% prompting hepatic disfunctions, use plasminogen and its variant in the present invention After being treated to subject, in patient's body PTA decline be expected will be improved significantly；3) imageological examination：Including belly Liver and gall spleen color ultrasound, CT or nuclear-magnetism, to understand hepatic injury recovery extent；4) tumor markers inspection, such as AFP, CA199, AFU etc.；5) liver impedance rheograph, to judge fibrosis and other recovery extents damaged.Moreover, it relates to make Subject is carried out with plasminogen and its variant in therapeutic process and after treatment, the judgement of the therapeutic scheme security, Serum half-life, treatment half-life period including but not limited to subject, median toxic dose (TD50), median lethal dose (LD50) Counted, or to the various adverse events such as sensitivity response of generation is observed over the course for the treatment of or after treatment.

Product or medicine box

One embodiment of the invention is related to a kind of product or medicine box, and it includes and can be used to treat what is caused by diabetes Hepatic injury and its plasminogen of the present invention of associated conditions.The product preferably includes a container, label or package insert. Appropriate container has bottle, bottle, syringe etc..Container can be made up of various materials such as glass or plastics.The container contains Composition, the composition can effectively treat disease of the invention or illness and have sterile access port (such as described container can be Intravenous solution bag or bottle, it contains the stopper that can be penetrated by hypodermic needle).At least one of composition activity Agent is plasminogen/fibrinolysin.On the container or appended label illustrate the composition be used to treating it is of the present invention by Hepatic injury and its associated conditions that diabetes cause.The product can further include the second appearance containing pharmaceutically acceptable buffer solution The salt solution of device, such as phosphate-buffered, Ringer's mixture and glucose solution.It can further include from business and user Other materials needed for from the point of view of angle, including other buffer solutions, diluent, filtrate, pin and syringe.Additionally, the product Comprising the package insert with operation instruction, including for example indicate the user of the composition by plasminogen composition and The other medicines administered patient of the adjoint disease for the treatment of.

Brief description

The change of diabetic mice body weight after plasminogen is given of Fig. 1 display 24-25 week old.

The diabetic mice of Fig. 2 display 24-25 week old liver HE dyeing observation knot after continuous 15 days administration plasminogens Really.

Fig. 3 display 24-25 week old diabetic mice gave plasminogen at continuous 15 days after hepatic fibrosis protein immunization Dyeing microscopic examination observes result.

Fig. 4 display 24-25 week old diabetic mice gave plasminogen at continuous 31 days after changes of weight.

Liver HE dyeing observations are tied after the diabetic mice of Fig. 5 display 24-25 week old gave plasminogen at continuous 31 days Really.

Fig. 6 display 24-25 week old diabetic mice gave plasminogen at continuous 31 days after hepatic fibrosis protein immunization Dyeing observation result.

Liver F4/80 immunostainings are seen after the diabetic mice of Fig. 7 display 24-25 week old gave plasminogen at continuous 31 days Examine result.

Fig. 8 shows that 24-25 weeks diabetic mice gives serum glutamic pyruvic transminase (ALT) inspection after 31 days of PBS or plasminogen Survey result.

Fig. 9 shows that the 0th, 2,7 days carbon tetrachloride for giving plasminogen cause acute hepatic injury mice liver HE dyeing observations As a result.

Figure 10 shows plg^-/-Carbon tetrachloride causes acute hepatic injury mice to give plasminogen liver HE after 18,24,48 hours Dyeing observation result.

Figure 11 shows plg^-/-Carbon tetrachloride cause acute hepatic injury mice give plasminogen after 18,24,48 hours liver it is fine Fibrillarin immunostaining observes result.

The mouse plasminogen of Figure 12 display 5.0Gy X-ray radiations immunostaining of liver F 4/80 observation result after 10 days.

Figure 13 shows the hepatic fibrosis protein immunization dyeing after 7 days of 10mg/Kg cisplatin chemotherapy damage model mouse plasminogens Observation result.

Figure 14 shows plg^-/-Carbon tetrachloride causes acute hepatic injury mice to give plasminogen 18,24,48 hours and after 7 days Liver HE dyeing observation results.

Embodiment

Influence of the plasminogen of embodiment 1 to neurotrosis later stage diabetic mice body weight

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and give plasminogen group, often Each 5.Experiment starts the same day and is designated as packet of weighing in the 0th day, tests and starts within second day to plasminogen or PBS and be designated as the 1st day, Successive administration 15 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, to solvent PBS pairs The PBS of same volume is given according to group.Giving plasminogen the 0th, 4,7,11,16 natural gift another name body weight.Result is shown to fibrinolysin Former group and to solvent PBS control group in the 0th, 4,7,11,16 days body weight (Fig. 1) without significant difference, illustrate plasminogen to animal Body weight influence is little.

The plasminogen of embodiment 2 damages the protective effect of advanced liver tissue damage to Diabetic liver

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and give plasminogen group, often Each 5.Experiment starts the same day and is designated as packet of weighing in the 0th day, tests and starts within second day to plasminogen or PBS and be designated as the 1st day, Successive administration 15 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, to solvent PBS pairs The PBS of same volume is given according to group.Mouse was put to death at the 16th day and liver organization is taken in 10% neutral formalin fixer Hepatic tissue after fixed fixation in 24-48 hours through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Histotomy Thickness is 5 μm, section dewaxing rehydration and with haematoxylin and eosin stains (HE dyeing), and 1% hydrochloride alcohol breaks up, and ammoniacal liquor returns indigo plant, And alcohol serial dehydration mounting.

HE coloration results show that give solvent PBS control group, liver cell becomes in severe fat, and lipidosis, nucleus is squeezed To edge, cell is slight hydropic degeneration, liver rope is disorderly；To plasminogen group compared to vehicle control group, liver cell fat becomes Property mitigate, in mild fatty become, based on moderate hydropic degeneration.Illustrate the reparation that plasminogen can promote Diabetic liver to damage.

The plasminogen of embodiment 3 reduces diabetic mice hepatic tissue fibrin level

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and give plasminogen group, often Each 5.Experiment starts the same day and is designated as packet of weighing in the 0th day, tests and starts within second day to plasminogen or PBS and be designated as the 1st day, Successive administration 15 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, to solvent PBS pairs The PBS of same volume is given according to group.Mouse was put to death at the 16th day and liver organization is taken in 10% neutral formalin fixer It is fixed 24-48 hours.Liver organization after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Tissue is cut Piece thickness is 5 μm, is washed 1 time after section dewaxing rehydration.It is incubated 15 minutes with 3% hydrogen peroxide, is washed 2 times, every time 5 minutes. 10% normal sheep serum liquid (Vector laboratories, Inc., USA) is closed 1 hour；After time arrives, reject sheep blood serum Liquid, tissue is irised out with PAP.Rabbit anti-mouse fibrin (original) antibody (Abcam) 4 DEG C of overnight incubations, TBS washes 2 times, and every time 5 Minute.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and TBS is washed 2 times, every time 5 minutes.Tried by DAB Agent box (Vector laboratories, Inc., USA) is developed the color, and haematoxylin is redyed 30 seconds after washing 3 times, and flowing water is rinsed 5 minutes, Then TBS is washed 1 time.Serial dehydration is transparent and mounting, and section is observed under 200 times under the microscope.

Fibrinogen is fibrinous precursor, is deposited in the case of a fracture in tissue, as body to the one of damage Plant stress reaction, fibrinogen hydrolysis fibroblast cells^[12-14].Therefore can be using fibrinogen level as degree of injury One mark.

Research finds, to plasminogen group (Fig. 3 B) compared with to solvent PBS control group (Fig. 3 A), to plasminogen group The fibrinous level reduction of its liver organization of mouse, illustrates that plasminogen has the function of suppressing fibrin level deposition, Damage obtains a certain degree of reparation.

Influence of the plasminogen of embodiment 4 to diabetic mice body weight

24-25 week old db/db hero mouse 20, are randomly divided into two groups, to solvent PBS control group and give plasminogen group, often Group is each 10.Experiment starts the same day and is designated as packet of weighing in the 0th day, tests and starts within second day to plasminogen or PBS and be designated as the 1st My god, successive administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS is given Control group gives the PBS of same volume.In the 0th, 4,7,11,16,21,26,31 natural gift another name body weight.

Result shows, to plasminogen group and to solvent PBS control group in the 0th, 4,7,11,16,21,26,31 day body weight Without significant difference (Fig. 4), illustrate that plasminogen is little on the weight of animals influence.

The plasminogen of embodiment 5 damages the protective effect of advanced liver tissue damage to Diabetic liver

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and give plasminogen group, often Each 5.Experiment starts the same day and is designated as packet of weighing in the 0th day, tests and starts within second day to plasminogen or PBS and be designated as the 1st day, Successive administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, to solvent PBS pairs The PBS of same volume is given according to group.Mouse was put to death at the 32nd day and liver organization is taken in 10% neutral formalin fixer Hepatic tissue after fixed fixation in 24-48 hours through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Histotomy Thickness is 5 μm, section dewaxing rehydration and with haematoxylin and eosin stains (HE dyeing), and 1% hydrochloride alcohol breaks up, and ammoniacal liquor returns indigo plant, And alcohol serial dehydration mounting.

HE coloration results show, are in severe steatosis, lipidosis, fusion to solvent PBS control group (Fig. 5 A) liver Into big fat vacuole, nucleus is pushed to edge (↗), liver rope is disorderly, and sinus hepaticus narrows, and does not have quantity at liver rope not etc. Inflammatory stove (↑)；The denaturation of plasminogen group (Fig. 5 B) liver mild fatty is given, is damaged based on slight hydropic degeneration, plasmolysis (◥), is mainly distributed on the region in the middle of portal area and central vein, is involved relatively light around portal area and central vein, while can See slight cell infiltration at liver rope.Illustrate substantially to be repaired to the damage of liver after plasminogen.

The plasminogen of embodiment 6 reduces diabetic mice hepatic tissue fibrin level

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and give plasminogen group, often Group is each 5.Experiment starts the same day and is designated as packet of weighing in the 0th day, tests and starts within second day to plasminogen or PBS and be designated as the 1st My god, successive administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS is given Control group gives the PBS of same volume.Mouse was put to death at the 32nd day and liver organization is taken in 10% neutral formalin fixer In fix 24 hours.Liver organization after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Tissue is cut Piece thickness is 5 μm, is washed 1 time after section dewaxing rehydration.It is incubated 15 minutes with 3% hydrogen peroxide, is washed 2 times, every time 5 minutes. 10% normal sheep serum liquid (Vector laboratories, Inc., USA) is closed 1 hour；After time arrives, reject sheep blood serum Liquid, tissue is irised out with PAP.Rabbit anti-mouse fibrin (original) antibody (Abcam) 4 DEG C of overnight incubations, TBS washes 2 times, and every time 5 Minute.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and TBS is washed 2 times, every time 5 minutes.Tried by DAB Agent box (Vector laboratories, Inc., USA) is developed the color, and haematoxylin is redyed 30 seconds after washing 3 times, and flowing water is rinsed 5 minutes. Serial dehydration is transparent and mounting, and section is observed under 200 times under the microscope.

Research finds, to plasminogen group (Fig. 6 B) compared with to solvent PBS control group (Fig. 6 A), to plasminogen group The fibrinous level of its liver organization of mouse is substantially reduced, and illustrates that injection plasminogen can significantly reduce diabetic mice Fibrin deposition, reflects that plasminogen has notable repair function to the body injury of diabetic mice.

The plasminogen of embodiment 7 mitigates the inflammation of diabetic mice liver organization

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and give plasminogen group, often Group is each 5.Experiment starts the same day and is designated as packet of weighing in the 0th day, tests and starts within second day to plasminogen or PBS and be designated as the 1st My god, successive administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS is given Control group gives the PBS of same volume.To putting to death mouse after plasminogen 31 days and take liver organization in 10% neutral formal 24 hours are fixed in woods fixer.Liver organization after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out paraffin bag Bury.Histotomy thickness is 5 μm, is washed 1 time after section dewaxing rehydration.It is incubated 15 minutes with 3% hydrogen peroxide, washing 2 times, every time 5 minutes.10% normal sheep serum is closed 1 hour, and the time gets rid of serum deprivation after, and tissue is irised out with PAP.For the rabbit of F4/80 4 DEG C of overnight incubations of polyclonal antibody (Abcam), TBS is washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) two Anti- incubation at room temperature 1 hour, TBS is washed 2 times.Developed the color by DAB kits (Vector laboratories, Inc., USA), washing 3 Secondary rear haematoxylin is redyed 30 seconds, and flowing water is rinsed 5 minutes, and serial dehydration is transparent and mounting, and section is seen under 400 times under the microscope Examine.

F4/80 macrophage markers, can represent degree and the stage of inflammatory reaction.Result shows, to plasminogen Compared with to solvent PBS control group (Fig. 7 A), the F4/80 Positive Levels to plasminogen group mouse are substantially reduced group (Fig. 7 B), Illustrate to mitigate to liver organization degree of inflammation after plasminogen.Fig. 7 C are F4/80 SABC positive expression number quantitative analysis knots Really, substantially reduced to plasminogen group F4/80 expression quantity, and with significant difference, illustrate that injection plasminogen can be notable Promote the reparation of diabetic mice inflammation.

The plasminogen of embodiment 8 promotes the reparation that diabetic mice liver is damaged

25-28 week old db/db hero mouse 9, are randomly divided into two groups, to solvent PBS control group 3, to plasminogen group 6 Only.Experiment starts the same day and is designated as packet of weighing in the 0th day, and experiment starts to plasminogen or PBS and is designated as the 1st day, company for second day Continuous administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS control is given Group gives the PBS of same volume.Eyeball is plucked after 31 days to plasminogen and adopts whole blood, after 4 DEG C of 3500r/min centrifugations after serum precipitation 10 minutes, take supernatant and detected.Using glutamic-pyruvic transaminase detection kit, (bio-engineering research is built up in Nanjing for this experiment Institute, article No. C009-2), glutamic-pyruvic transaminase (ALT's) contains in Lai Shi colorimetric methods (Reitman-Frankel) detection serum Amount.

Glutamic-pyruvic transaminase is an important indicator of liver health state^[15,16], the normal reference value area of glutamic-pyruvic transaminase Between be 9~50U/L.Testing result shows that the content to solvent PBS control group serum alt is significantly higher than normal physiological index, And have been restored to internal normal level to plasminogen group, and be significantly lower than to solvent PBS pairs to plasminogen group According to group, and with significant difference (Fig. 8).Illustrate in advanced diabetes model mice, injection plasminogen can be repaiied effectively Multiple hepatic injury.

Protective effect of the plasminogen of embodiment 9 to acute liver poisoning liver

7-8 week old plg^+/+Mouse 18, male and female are not limited, and are randomly divided into two groups, respectively to solvent PBS control group and fibre Lyase original administration group, every group 9.Two groups of mouse are administered to through abdominal cavity by 0.5mL/kg body weight and give carbon tetrachloride, continuously give two My god, set up acute hepatic injury model^[17,18].Carbon tetrachloride need to be diluted before with corn oil, the volume ratio 1 of the former with the latter: 7.The modeling same day is the 0th day, and the 1st day starts to plasminogen or PBS.Daily 1mg/0.1mL/ is pressed to plasminogen group mouse Pcs/day plasminogen is given, the PBS of same volume, successive administration 7 days are given to solvent PBS control group.In the 0th, 2,7 natural gift Two groups of each 3 execution of mouse are not taken, and anatomic observation records liver situation, and then liver organization is fixed in 10% neutral formalin 24-48 hours is fixed in liquid.Liver organization after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Group Slice thickness is knitted for 5 μm, section dewaxing rehydration and with haematoxylin and eosin stains (HE dyeing), 1% hydrochloride alcohol breaks up, ammoniacal liquor Indigo plant, and alcohol serial dehydration mounting are returned, is observed under 200 times under the microscope.

HE coloration results show, to solvent PBS control group (Fig. 9 A-C) and the liver to plasminogen group (Fig. 9 D-F) mouse Dirty 0th day main based on piecemeal necrosis around central vein, necrotic zone karyon fragmentation, the light dye of endochylema, and other are not downright bad Area also there occurs moderate hydropic degeneration, edema；Central vein expansion in 2nd day, liver cell structure disturbance, a small amount of inflammatory cell Infiltration, two groups and no significant difference.But at the 7th day, give solvent PBS control group still visible a small amount of degeneration of liver cells, cell Mild edema, liver rope is disorderly, and hepatic sinusoid narrows, and the slight cell infiltration around portal area, and to the liver of plasminogen group Dirty to return to normal the red dye of endochylema, liver rope rule, sinus hepaticus is clear.Illustrate that plasminogen can promote the reparation of hepatic injury.

Protective effect of the plasminogen of embodiment 10 to acute liver poisoning liver

7-11 week old plg^-/-Male mice 18, is randomly divided into two groups, respectively to solvent PBS control group and fibrinolysin Former administration group, every group 9.Two groups of mouse are administered to through abdominal cavity by 0.5mL/kg body weight and give carbon tetrachloride, single treatment, set up anxious Property liver injury model^[17,^18].Carbon tetrachloride need to be diluted before with corn oil, the volume ratio 1 of the former with the latter:7.Modeling gives Plasminogen or solvent PBS are given in into half an hour after.To plasminogen group mouse fibre is given by daily 1mg/0.1mL/ pcs/day Lyase is former, and the PBS of same volume, successive administration 2 days are given to solvent PBS control group.18th, 24,48 small time-division upon administration Two groups of each 3 execution of mouse are not taken, and anatomic observation records liver situation, and then liver organization is fixed in 10% neutral formalin 24-48 hours is fixed in liquid.Liver organization after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Group Slice thickness is knitted for 5 μm, section dewaxing rehydration and with haematoxylin and eosin stains (HE dyeing), 1% hydrochloride alcohol breaks up, ammoniacal liquor Indigo plant, and alcohol serial dehydration mounting are returned, is observed under 200 times under the microscope.

Result shows, different degrees of necrosis is presented in 18h, 24h, 48h to solvent PBS control group (Figure 10 A-C), 18h, 24h have occurred that bridging necrosis, karyorrhexis, the light dye of endochylema, and damage continuous adding based on sheet necrosis during 48h Play, is mainly distributed on around central vein, necrotic area moderate cell infiltration (↓), and necrosis is relatively light around portal area, with slight water Based on sample denaturation, slight cell infiltration, slight bile duct proliferation (◥) are accompanied by；To plasminogen group (Figure 10 D-F) 18h, 24h, 48h do not occur obvious necrosis compared to control group, damage based on slight hydropic degeneration, are distributed in portal area week Enclose, and liver cell is not involved around central vein, and during 24h than 18h take a favorable turn, hydropic degeneration mitigates, around central vein Slight hepatic cell steatosis, the light dye of endochylema, accompanies by slight cell infiltration.Illustrate that plasminogen can promote plg^-/-It is anxious Property the reparation that damages of liver injury model mouse liver.

The plasminogen of embodiment 11 mitigates acute hepatic injury model mouse liver tissue fibrin deposition

7-11 week old plg^-/-Male mice 18, is randomly divided into two groups, respectively to solvent PBS control group and fibrinolysin Former administration group, every group 9.Two groups of mouse press 0.5mL/kg body weight through intraperitoneal injection carbon tetrachloride, and single treatment sets up Acute Hepatic Damage model^[17,18].Carbon tetrachloride need to be diluted before with corn oil, the volume ratio 1 of the former with the latter:7.After the completion of modeling is given Plasminogen or solvent PBS are given in half an hour.To plasminogen group mouse fibrinolysin is given by daily 1mg/0.1mL/ pcs/day Original, the PBS of same volume, successive administration 2 days are given to solvent PBS control group.Take respectively within the 18th, 24,48 hour upon administration Two groups of each 3 execution of mouse, anatomic observation record liver situation, then liver organization is in 10% neutral formalin fixer It is fixed 24-48 hours.Liver organization after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Tissue is cut Piece thickness is 5 μm, is washed 1 time after section dewaxing rehydration.It is incubated 15 minutes with 3% hydrogen peroxide, is washed 2 times, every time 5 minutes. 10% normal sheep serum liquid (Vector laboratories, Inc., USA) is closed 1 hour；After time arrives, reject sheep blood serum Liquid, tissue is irised out with PAP.Rabbit anti-mouse fibrin (original) antibody (Abcam) 4 DEG C of overnight incubations, TBS washes 2 times, and every time 5 Minute.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and TBS is washed 2 times, every time 5 minutes.Tried by DAB Agent box (Vector laboratories, Inc., USA) is developed the color, and haematoxylin is redyed 30 seconds after washing 3 times, and flowing water is rinsed 5 minutes. Serial dehydration is transparent and mounting, and section is observed under 200 times under the microscope.

Result shows that the 18th, 24,48 hour three time point gave plasminogen group (Figure 11 D-F) fibrinous positive Coloring is all significantly shallower than and gives solvent PBS control group (Figure 11 A-C), and the fibrinous coloring of extension over time also have by The shallow trend of gradual change.Illustrate that injection plasminogen can reduce fibrinous deposition, promote the reparation of hepatic injury.

The plasminogen of embodiment 12 promotes the reparation of 5.0GyX ray radiation murine inflammations

This experiment is randomly divided into two groups using the male C57 mouse 10 of 6-8 week old health, to solvent PBS control group and Give plasminogen group, every group each 5.After the completion of packet, radiation insult model is set up, pressed using linear accelerator 6MV X-rays To mouse systemic single uniform irradiation, absorbed dose rate 2.0Gy/min, absorbed dose of radiation is 5.0Gy (irradiation 2.5 minutes) to 5.0Gy. After setting up model, plasminogen is given in 3 hours.Experiment starts the same day for the 0th day weighs in and be grouped, and the 1st day starts Radiation treatment simultaneously gives plasminogen or solvent PBS, and administration phase is to carry out withdrawal observation 11 days to animal after the completion of being administered within 10 days, Whole experiment periods are 21 days.1mg/0.1mL/ pcs/day is pressed to plasminogen group to be administered through tail vein injection, give solvent PBS control Group gives the PBS of same volume.Put to death to dissect mouse and take liver at the 21st day and consolidate in 10% neutral formalin fixer Determine 24-48 hours.Liver organization after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Histotomy Thickness is 5 μm, is washed 1 time after section dewaxing rehydration.Tris-EDTA is repaired 30 minutes, and water is softly rushed after room temperature is cooled down 20 minutes Wash.It is incubated 15 minutes with 3% hydrogen peroxide, tissue is irised out with PAP.10% normal sheep serum (Vector Laboratories, Inc., USA) close 1 hour；After time arrives, reject sheep blood serum liquid.Rabbit anti-mouse F4/80 antibody (Abcam) 4 DEG C of overnight incubations, TBS is washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, TBS was washed 2 times, every time 5 minutes.Developed the color by DAB kits (Vector laboratories, Inc., USA), washing 3 Secondary rear haematoxylin is redyed 30 seconds, and flowing water is rinsed 5 minutes.Serial dehydration is transparent and mounting, and section is seen under 200 times under the microscope Examine.

F4/80 Showed by immune group result, to the small of solvent PBS control group (Figure 12 A) after 5.0Gy x-ray bombardment modelings The expression quantity of mouse macrophage mark is illustrated to after plasminogen, animal's liver tissue higher than giving plasminogen group (Figure 12 B) Inflammation significantly mitigate.

The plasminogen of embodiment 13 reduces the deposition of cisplatin chemotherapy damage model mouse liver tissue fibrin

The male C57 mouse 10 of 8-9 week old health, are randomly divided into two groups, to solvent PBS control group and to plasminogen Group, every group each 5.After the completion of packet, chemotherapy damage model is set up, by 10mg/Kg body weight single intraperitoneal injection cis-platinums.Set up mould After type, plasminogen is given through tail vein injection by 1mg/ pcs/day to plasminogen group, given to solvent PBS control group identical The PBS of volume.Experiment starts the same day for the 0th day weighs in and be grouped, and the 1st day starts intraperitoneal injection cis-platinum modeling, 3 after modeling Plasminogen or solvent PBS are given in hour, administration phase is 7 days.Put to death mouse within 8th day, take liver in 10% neutral formal 24-48 hours is fixed in woods fixer.Liver organization after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out paraffin Embedding.Histotomy thickness is 5 μm, is washed 1 time after section dewaxing rehydration.Citric acid is repaired 30 minutes, and room temperature is cooled down 10 minutes Water is softly rinsed afterwards.It is incubated 15 minutes with 3% hydrogen peroxide, tissue is irised out with PAP.10% normal sheep serum (Vector Laboratories, Inc., USA) close 1 hour；After time arrives, reject sheep blood serum liquid.Rabbit anti-mouse fibrin antibody (Abcam) 4 DEG C of overnight incubations, TBS is washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, TBS was washed 2 times, every time 5 minutes.Developed the color by DAB kits (Vector laboratories, Inc., USA), washing 3 Secondary rear haematoxylin is redyed 30 seconds, and flowing water is rinsed 5 minutes.Serial dehydration is transparent and mounting, and section is seen under 200 times under the microscope Examine.

Result shows, is coloured to the fibrin positive in solvent PBS control group (Figure 13 A) liver organization and be substantially deeper than fibrinolytic Proenzyme administration group (Figure 13 B).Illustrate that plasminogen can substantially reduce the fibrin deposited in the liver organization of damage, table Bright plasminogen can promote the reparation of the hepar damnification caused by chemotherapeutic drugs Cisplatin.

Protective effect of the plasminogen of embodiment 14 to acute liver poisoning liver

7-11 week old plg-/- male mice 6, is randomly divided into two groups, respectively to solvent PBS control group and fibrinolysin Former administration group, every group 3.Two groups of mouse are administered to through abdominal cavity by 0.5mL/kg body weight and give carbon tetrachloride, single treatment, set up anxious Property liver injury model^[17,18].Carbon tetrachloride need to be diluted before with corn oil, the volume ratio 1 of the former with the latter:7.Modeling gives Plasminogen or solvent PBS are given in into half an hour after.To plasminogen group mouse fibre is given by daily 1mg/0.1mL/ pcs/day Lyase is former, and the PBS of same volume, successive administration 7 days are given to solvent PBS control group.Mouse, anatomic observation were put to death at the 8th day Liver situation is recorded, then liver organization fixes 24-48 hours in 10% neutral formalin fixer.Liver after fixation Tissue through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Histotomy thickness is 5 μm, and section dewaxing rehydration is simultaneously With haematoxylin and eosin stains (HE dyeing), the differentiation of 1% hydrochloride alcohol, ammoniacal liquor returns indigo plant, and alcohol serial dehydration mounting, micro- Observed under lower 200 times of mirror.

Result shows that give solvent PBS control group (Figure 14 A), liver central vein expansion, endothelial cell necrosis, center is quiet The equal occurrence of large-area focal necrosis of surrounding liver cell of arteries and veins, karyorrhexis contaminates deeply, and there is slight water in other regions not necrosed Sample is denatured, and edema, endochylema is bright, accompanies by the slight cell infiltration in necrotic area；Plasminogen group (Figure 14 B) is given, liver is not There is obvious necrosis, damage based on slight hydropic degeneration, there is a small amount of liver cell eosinophilic cytoplasmic to strengthen, red dye.To fibre The former group of lyase is damaged substantially to be lighter than and gives solvent PBS control group, illustrates that plasminogen can promote plg-/- acute hepatic injury model The reparation that mouse liver is damaged.

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[2]Tim CMA Schreuder,Bart J Verwer,Carin MJ van Nieuwkerk,Chris JJ Mulder,

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[3]Wiman,B.and Wallen,P.(1975).Structural relationship between" glutamic acid"and"lysine"forms of human plasminogen and their interaction with the NH2-terminal activation peptide as studied by affinity chromatography.Eur.J.Biochem.50,489-494.

[4]Saksela,O.and Rifkin,D.B.(1988).Cell-associated plasminogen activation:regulation and physiological functions.Annu.Rev.Cell Biol.4,93-126

[5]Raum,D.,Marcus,D.,Alper,C.A.,Levey,R.,Taylor,P.D.,and Starzl,T.E. (1980).Synthesis of human plasminogen by the liver.Science 208,1036-1037

[6]Wallén P(1980).Biochemistry of plasminogen.In Fibrinolysis,Kline DL and Reddy KKN,eds.(Florida:CRC.

[7]Sottrup-Jensen,L.,Zajdel,M.,Claeys,H.,Petersen,T.E.,and Magnusson, S.(1975).Amino-acid sequence of activation cleavage site in plasminogen: homology with"pro"part of prothrombin.Proc.Natl.Acad.Sci.U.S.A 72,2577-2581.

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Sequence table

<110>Shenzhen Co., Ltd of Rui Jian life sciences institute

<120>A kind of method for preventing and treating liver tissue injury and its associated conditions

<130> PCK02773

<160> 14

<170> PatentIn version 3.5

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<213>Natural plasminogen (Glu-PLG, Glu- plasminogen) nucleotide sequence of signal peptide is not contained

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ggtccctggt gctacacgac aaatccaaga aaactttacg actactgtga tgtccctcag 1620

tgtgcggccc cttcatttga ttgtgggaag cctcaagtgg agccgaagaa atgtcctgga 1680

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agtggaggtc ctctggtttg cttcgagaag gacaaataca ttttacaagg agtcacttct 2280

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<213>Natural plasminogen (Glu-PLG, Glu- plasminogen) amino acid sequence of signal peptide is not contained

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Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser

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Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala

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Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His

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Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser

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Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr

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Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met

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Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser

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Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu

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Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp

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Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu

145 150 155 160

Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly

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Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser

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Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys

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Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro

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Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile

225 230 235 240

Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys

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Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val

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Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His

275 280 285

Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr

290 295 300

Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn

305 310 315 320

Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser

325 330 335

Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr

340 345 350

Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly

355 360 365

Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser

370 375 380

Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala

385 390 395 400

Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro

405 410 415

Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu

420 425 430

Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val

435 440 445

Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe

450 455 460

Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly

465 470 475 480

Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile

485 490 495

Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys

500 505 510

Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn

515 520 525

Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro

530 535 540

Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly

545 550 555 560

Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln

565 570 575

Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu

580 585 590

Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser

595 600 605

Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val

610 615 620

Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu

625 630 635 640

Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala

645 650 655

Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr

660 665 670

Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr

675 680 685

Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val

690 695 700

Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val

705 710 715 720

Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser

725 730 735

Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys

740 745 750

Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro

755 760 765

Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile

770 775 780

Glu Gly Val Met Arg Asn Asn

785 790

<210> 3

<211> 2433

<212> DNA

<213>Natural plasminogen containing signal peptide（From swiss prot）Nucleotide sequence

<400> 3

atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagg tcaaggagag 60

cctctggatg actatgtgaa tacccagggg gcttcactgt tcagtgtcac taagaagcag 120

ctgggagcag gaagtataga agaatgtgca gcaaaatgtg aggaggacga agaattcacc 180

tgcagggcat tccaatatca cagtaaagag caacaatgtg tgataatggc tgaaaacagg 240

aagtcctcca taatcattag gatgagagat gtagttttat ttgaaaagaa agtgtatctc 300

tcagagtgca agactgggaa tggaaagaac tacagaggga cgatgtccaa aacaaaaaat 360

ggcatcacct gtcaaaaatg gagttccact tctccccaca gacctagatt ctcacctgct 420

acacacccct cagagggact ggaggagaac tactgcagga atccagacaa cgatccgcag 480

gggccctggt gctatactac tgatccagaa aagagatatg actactgcga cattcttgag 540

tgtgaagagg aatgtatgca ttgcagtgga gaaaactatg acggcaaaat ttccaagacc 600

atgtctggac tggaatgcca ggcctgggac tctcagagcc cacacgctca tggatacatt 660

ccttccaaat ttccaaacaa gaacctgaag aagaattact gtcgtaaccc cgatagggag 720

ctgcggcctt ggtgtttcac caccgacccc aacaagcgct gggaactttg tgacatcccc 780

cgctgcacaa cacctccacc atcttctggt cccacctacc agtgtctgaa gggaacaggt 840

gaaaactatc gcgggaatgt ggctgttacc gtgtccgggc acacctgtca gcactggagt 900

gcacagaccc ctcacacaca taacaggaca ccagaaaact tcccctgcaa aaatttggat 960

gaaaactact gccgcaatcc tgacggaaaa agggccccat ggtgccatac aaccaacagc 1020

caagtgcggt gggagtactg taagataccg tcctgtgact cctccccagt atccacggaa 1080

caattggctc ccacagcacc acctgagcta acccctgtgg tccaggactg ctaccatggt 1140

gatggacaga gctaccgagg cacatcctcc accaccacca caggaaagaa gtgtcagtct 1200

tggtcatcta tgacaccaca ccggcaccag aagaccccag aaaactaccc aaatgctggc 1260

ctgacaatga actactgcag gaatccagat gccgataaag gcccctggtg ttttaccaca 1320

gaccccagcg tcaggtggga gtactgcaac ctgaaaaaat gctcaggaac agaagcgagt 1380

gttgtagcac ctccgcctgt tgtcctgctt ccagatgtag agactccttc cgaagaagac 1440

tgtatgtttg ggaatgggaa aggataccga ggcaagaggg cgaccactgt tactgggacg 1500

ccatgccagg actgggctgc ccaggagccc catagacaca gcattttcac tccagagaca 1560

aatccacggg cgggtctgga aaaaaattac tgccgtaacc ctgatggtga tgtaggtggt 1620

ccctggtgct acacgacaaa tccaagaaaa ctttacgact actgtgatgt ccctcagtgt 1680

gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 1740

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 1800

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 1860

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 1920

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 1980

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 2040

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 2100

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 2160

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 2220

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 2280

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 2340

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 2400

acttggattg agggagtgat gagaaataat taa 2433

<210> 4

<211> 810

<212> PRT

<213>Natural plasminogen containing signal peptide（From swiss prot）Amino acid sequence

<400> 4

Met Glu His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser

1 5 10 15

Gly Gln Gly Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser

20 25 30

Leu Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu

35 40 45

Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe

50 55 60

Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg

65 70 75 80

Lys Ser Ser Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys

85 90 95

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

100 105 110

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

115 120 125

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

130 135 140

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

145 150 155 160

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

165 170 175

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

180 185 190

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

195 200 205

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

210 215 220

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

225 230 235 240

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

245 250 255

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

260 265 270

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala

275 280 285

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

290 295 300

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

305 310 315 320

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

325 330 335

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

340 345 350

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

355 360 365

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

370 375 380

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

385 390 395 400

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

405 410 415

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

420 425 430

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

435 440 445

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

450 455 460

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

465 470 475 480

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

485 490 495

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

500 505 510

His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys

515 520 525

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

530 535 540

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

545 550 555 560

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

565 570 575

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

580 585 590

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

595 600 605

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

610 615 620

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

625 630 635 640

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

645 650 655

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

660 665 670

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

675 680 685

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

690 695 700

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

705 710 715 720

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

725 730 735

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

740 745 750

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

755 760 765

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

770 775 780

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

785 790 795 800

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

805 810

<210> 5

<211> 2145

<212> DNA

<213> LYS77-PLG（Lys- plasminogens）Nucleotide sequence

<400> 5

aaagtgtatc tctcagagtg caagactggg aatggaaaga actacagagg gacgatgtcc 60

aaaacaaaaa atggcatcac ctgtcaaaaa tggagttcca cttctcccca cagacctaga 120

ttctcacctg ctacacaccc ctcagaggga ctggaggaga actactgcag gaatccagac 180

aacgatccgc aggggccctg gtgctatact actgatccag aaaagagata tgactactgc 240

gacattcttg agtgtgaaga ggaatgtatg cattgcagtg gagaaaacta tgacggcaaa 300

atttccaaga ccatgtctgg actggaatgc caggcctggg actctcagag cccacacgct 360

catggataca ttccttccaa atttccaaac aagaacctga agaagaatta ctgtcgtaac 420

cccgataggg agctgcggcc ttggtgtttc accaccgacc ccaacaagcg ctgggaactt 480

tgtgacatcc cccgctgcac aacacctcca ccatcttctg gtcccaccta ccagtgtctg 540

aagggaacag gtgaaaacta tcgcgggaat gtggctgtta ccgtgtccgg gcacacctgt 600

cagcactgga gtgcacagac ccctcacaca cataacagga caccagaaaa cttcccctgc 660

aaaaatttgg atgaaaacta ctgccgcaat cctgacggaa aaagggcccc atggtgccat 720

acaaccaaca gccaagtgcg gtgggagtac tgtaagatac cgtcctgtga ctcctcccca 780

gtatccacgg aacaattggc tcccacagca ccacctgagc taacccctgt ggtccaggac 840

tgctaccatg gtgatggaca gagctaccga ggcacatcct ccaccaccac cacaggaaag 900

aagtgtcagt cttggtcatc tatgacacca caccggcacc agaagacccc agaaaactac 960

ccaaatgctg gcctgacaat gaactactgc aggaatccag atgccgataa aggcccctgg 1020

tgttttacca cagaccccag cgtcaggtgg gagtactgca acctgaaaaa atgctcagga 1080

acagaagcga gtgttgtagc acctccgcct gttgtcctgc ttccagatgt agagactcct 1140

tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcgaccact 1200

gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 1260

actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 1320

gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 1380

gtccctcagt gtgcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 1440

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 1500

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 1560

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 1620

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 1680

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 1740

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 1800

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 1860

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1920

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1980

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 2040

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 2100

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 2145

<210> 6

<211> 714

<212> PRT

<213> LYS77-PLG（Lys- plasminogens）Amino acid sequence

<400> 6

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

1 5 10 15

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

20 25 30

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

35 40 45

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

50 55 60

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

65 70 75 80

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

85 90 95

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

100 105 110

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

115 120 125

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

130 135 140

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

145 150 155 160

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

165 170 175

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala

180 185 190

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

195 200 205

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

210 215 220

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

225 230 235 240

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

245 250 255

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

260 265 270

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

275 280 285

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

290 295 300

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

305 310 315 320

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

325 330 335

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

340 345 350

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

355 360 365

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

370 375 380

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

385 390 395 400

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

405 410 415

His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys

420 425 430

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

435 440 445

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

450 455 460

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

465 470 475 480

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

485 490 495

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

500 505 510

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

515 520 525

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

530 535 540

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

545 550 555 560

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

565 570 575

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

580 585 590

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

595 600 605

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

610 615 620

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

625 630 635 640

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

645 650 655

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

660 665 670

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

675 680 685

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

690 695 700

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

705 710

<210> 7

<211> 1245

<212> DNA

<213> delta-plg（Delta- plasminogens）Nucleotide sequence

<400> 7

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 540

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 600

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 660

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 720

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 780

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 840

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 900

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 960

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1020

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1080

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 1140

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 1200

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 1245

<210> 8

<211> 414

<212> PRT

<213> delta-plg（Delta- plasminogens）Amino acid sequence

<400> 8

Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser

1 5 10 15

Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala

20 25 30

Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His

35 40 45

Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser

50 55 60

Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr

65 70 75 80

Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met

85 90 95

Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser

100 105 110

Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu

115 120 125

Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp

130 135 140

Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu

145 150 155 160

Glu Cys Glu Glu Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val

165 170 175

Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His

180 185 190

Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met

195 200 205

His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala

210 215 220

Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile

225 230 235 240

Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile

245 250 255

Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu

260 265 270

Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala

275 280 285

Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe

290 295 300

Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu

305 310 315 320

Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr

325 330 335

Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His

340 345 350

Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu

355 360 365

Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp

370 375 380

Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val

385 390 395 400

Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

405 410

<210> 9

<211> 1104

<212> DNA

<213> Mini-plg（Small plasminogen）Nucleotide sequence

<400> 9

gtcaggtggg agtactgcaa cctgaaaaaa tgctcaggaa cagaagcgag tgttgtagca 60

cctccgcctg ttgtcctgct tccagatgta gagactcctt ccgaagaaga ctgtatgttt 120

gggaatggga aaggataccg aggcaagagg gcgaccactg ttactgggac gccatgccag 180

gactgggctg cccaggagcc ccatagacac agcattttca ctccagagac aaatccacgg 240

gcgggtctgg aaaaaaatta ctgccgtaac cctgatggtg atgtaggtgg tccctggtgc 300

tacacgacaa atccaagaaa actttacgac tactgtgatg tccctcagtg tgcggcccct 360

tcatttgatt gtgggaagcc tcaagtggag ccgaagaaat gtcctggaag ggttgtaggg 420

gggtgtgtgg cccacccaca ttcctggccc tggcaagtca gtcttagaac aaggtttgga 480

atgcacttct gtggaggcac cttgatatcc ccagagtggg tgttgactgc tgcccactgc 540

ttggagaagt ccccaaggcc ttcatcctac aaggtcatcc tgggtgcaca ccaagaagtg 600

aatctcgaac cgcatgttca ggaaatagaa gtgtctaggc tgttcttgga gcccacacga 660

aaagatattg ccttgctaaa gctaagcagt cctgccgtca tcactgacaa agtaatccca 720

gcttgtctgc catccccaaa ttatgtggtc gctgaccgga ccgaatgttt catcactggc 780

tggggagaaa cccaaggtac ttttggagct ggccttctca aggaagccca gctccctgtg 840

attgagaata aagtgtgcaa tcgctatgag tttctgaatg gaagagtcca atccaccgaa 900

ctctgtgctg ggcatttggc cggaggcact gacagttgcc agggtgacag tggaggtcct 960

ctggtttgct tcgagaagga caaatacatt ttacaaggag tcacttcttg gggtcttggc 1020

tgtgcacgcc ccaataagcc tggtgtctat gttcgtgttt caaggtttgt tacttggatt 1080

gagggagtga tgagaaataa ttaa 1104

<210> 10

<211> 367

<212> PRT

<213> Mini-plg（Small plasminogen）Amino acid sequence

<400> 10

Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala

1 5 10 15

Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr

20 25 30

Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly

35 40 45

Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala

50 55 60

Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg

65 70 75 80

Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly

85 90 95

Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys

100 105 110

Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln

115 120 125

Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala

130 135 140

His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly

145 150 155 160

Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr

165 170 175

Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val

180 185 190

Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu

195 200 205

Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala

210 215 220

Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro

225 230 235 240

Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys

245 250 255

Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu

260 265 270

Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg

275 280 285

Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly

290 295 300

His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro

305 310 315 320

Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser

325 330 335

Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg

340 345 350

Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

355 360 365

<210> 11

<211> 750

<212> DNA

<213> Micro-plg（Fibrillin lyase is former）Nucleotide sequence

<400> 11

gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 60

gtaggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 120

tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 180

cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 240

gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 300

acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 360

atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 420

actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 480

cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 540

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 600

ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 660

cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 720

tggattgagg gagtgatgag aaataattaa 750

<210> 12

<211> 249

<212> PRT

<213> Micro-plg（Fibrillin lyase is former）Amino acid sequence

<400> 12

Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys

1 5 10 15

Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro

20 25 30

Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly

35 40 45

Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu

50 55 60

Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln

65 70 75 80

Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu

85 90 95

Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser

100 105 110

Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro

115 120 125

Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly

130 135 140

Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu

145 150 155 160

Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly

165 170 175

Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr

180 185 190

Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys

195 200 205

Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala

210 215 220

Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr

225 230 235 240

Trp Ile Glu Gly Val Met Arg Asn Asn

245

<210> 13

<211> 684

<212> DNA

<213>Serine protease（Structure）The nucleotide sequence in domain

<400> 13

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 60

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 120

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 180

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 240

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 300

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 360

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 420

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 480

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 540

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 600

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 660

acttggattg agggagtgat gaga 684

<210> 14

<211> 228

<212> PRT

<213>Serine protease（Structure）The amino acid sequence in domain

<400> 14

Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val

1 5 10 15

Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile

20 25 30

Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro

35 40 45

Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn

50 55 60

Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu

65 70 75 80

Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val

85 90 95

Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val

100 105 110

Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln

115 120 125

Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile

130 135 140

Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln

145 150 155 160

Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys

165 170 175

Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr

180 185 190

Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn

195 200 205

Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu

210 215 220

Gly Val Met Arg

225

Claims

1. plasminogen prepare prevention and/or treatment subject's liver tissue injury and its associated conditions medicine in purposes.

2. the purposes of claim 1, wherein the liver tissue injury and its associated conditions are the liver that radiation or chemical substance cause Damage and its associated conditions.

3. the purposes of claim 1, wherein the liver tissue injury and its associated conditions are toxic liver injury and its related diseases Disease.

4. the purposes of claim 1, wherein the liver tissue injury and its associated conditions are diabetic keratopathy hepatic injury and its correlation Illness.

5. purposes according to claim 4, wherein the diabetic keratopathy hepatic injury and its associated conditions are caused by diabetes Big blood vessel, thin vessels, microangiopathies cause.

6. the purposes of any one of claim 1-5, wherein the liver tissue injury and its associated conditions are led including liver tissue injury The dysfunction of liver of cause, liver enzyme are abnormal, uncomfortable liver area and tenderness, hepatomegaly, splenomegaly, hepatosplenomegaly, hepatitis, fatty liver, Cholangitis, cirrhosis, hepatonecrosis and liver cancer.

7. according to the purposes of claim 1-6, wherein the plasminogen has at least with sequence 2,6,8,10 or 12 80%th, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is fibrinolysin Former activity.

8. according to the purposes of claim any one of 1-7, wherein the plasminogen is comprising activities of endothelial tissue plasminogen piece Section and still have plasminogen activity protein.

9. according to the purposes of claim any one of 1-8, wherein the plasminogen is selected from Glu- plasminogens, Lys- Plasminogen, small plasminogen, fibrillin lyase original, δ (delta)-plasminogen or its any group Close.

10. according to the purposes of claim any one of 1-9, wherein the plasminogen can combine with one or more other medicines Using.