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CN106344611A - Nutrition compound and application in preparing drug for promoting stem cell proliferation - Google Patents

Nutrition compound and application in preparing drug for promoting stem cell proliferation Download PDF

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Publication number
CN106344611A
CN106344611A CN201610720042.1A CN201610720042A CN106344611A CN 106344611 A CN106344611 A CN 106344611A CN 201610720042 A CN201610720042 A CN 201610720042A CN 106344611 A CN106344611 A CN 106344611A
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China
Prior art keywords
vitamin
stem cells
application
stem cell
alimentation composition
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CN201610720042.1A
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Chinese (zh)
Inventor
吴文国
贾莉
杨佩满
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Dalian Jinfu Biological Technology Development Co Ltd
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Dalian Jinfu Biological Technology Development Co Ltd
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Priority to CN201610720042.1A priority Critical patent/CN106344611A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
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    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
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    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
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    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4415—Pyridoxine, i.e. Vitamin B6
    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7135—Compounds containing heavy metals
    • A61K31/714—Cobalamins, e.g. cyanocobalamin, i.e. vitamin B12
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
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Abstract

The invention discloses a nutrition compound and application in preparing a drug for promoting stem cell proliferation. The nutrition compound is prepared from the following materials in proportion: lysine, methionine, phenylalanine, threonine, tryptophan, arginine, histidine, glycine, aspartic acid, leucine, isoleucine, valine, serine, glutamine, taurine, orotic acid, nucleotide, Vitamin A, Vitamin D, Vitamin B2, Vitamin B6, Vitamin B12, niacin, folic acid, Vitamin C, iron, zinc, manganese, copper, selenium, chromium, potassium, calcium, magnesium, inositol and granulesten. The nutrition compound has the effects of boosting the proliferation of hematopoietic stem cells, liver stem cells, mesenchymal stem cells, kidney stem cells and gastric mucosa stem cells. The nutrition compound can be used for preparing the drug for promoting stem cell proliferation, is free from toxic and side effect and can avoid various problems caused by introduction of exogenous stem cells for treating diseases.

Description

Alimentation composition and the application in preparation promotion stem cells hyperplasia medicine
Technical field
The present invention relates to a kind of alimentation composition and purposes, especially one kind have promotion hematopoietic stem cell, mesenchyme is done The alimentation composition of five kinds of stem cells hyperplasias such as cell and the application in preparation stem cells hyperplasia medicine.
Background technology
With the continuous development of stem cell biology research, the transplantation treatment with stem cell as seed cell will carry for patient Carry out new hope.But, under conditions of body macroscopic view and micro environment do not obtain any improvement, its own is original accordingly to do In the state of cell is still in " dormancy ", the stem cell conveying relying solely on foreign aid's property is it is clear that difficult to reach expected purpose.With When to realize for stem cell becoming medicine and also have many key issues to need to solve, for example, how to keep stem cell through body Differentiation capability after outer large-scale culture, inherited characteristic, oncogenicity and stability；Set up the external biological related to drug action Learn evaluation methodology；Screening keeps the stable pharmaceutical formulation of living cells；Select dosage form and packaging material；Specification storage, cold chain Traffic condition；Set up technical standard and the quality evaluation system of stem cell products；Grope Preclinical Drug efficiency evaluation system, Safety assessment system etc., cumbersome.
The Chinese invention patent of Patent No. 200710012788.8 discloses a kind of " promotion hemopoietic stem cell proliferation and blood The alimentation composition of Lactoferrin synthesis ".The raw material of this alimentation composition and weight ratio are as follows: nucleotide 90 ~ 110, arginase 12 0 ~ 30th, lysine 20 ~ 30, cysteine 10 ~ 20, glycine 25 ~ 35, histidine 20 ~ 30, lecithin 110 ~ 130, cephalin 50 ~ 70th, vitamin e 0.4 ~ 0.6, vitamin c 5 ~ 7, Folic Acid 0.02 ~ 0.04, vitamin b20.05 ~ 0.07, vitamin b60.07~ 0.08th, vitamin b120.0001 ~ 0.0002, ferrum 0.5 ~ 1.5, zinc 0.5 ~ 0.7, manganese 0.4 ~ 0.6, lycium barbarum polysaccharide 14 ~ 16, Fructus Vitis viniferae Seed extract 14 ~ 16.Blood hemoglobin (hb), erythrocyte (rbc), leukocyte (wbc) and platelet (plt) can be significantly improved Quantity；The propagation of marrow hemopoietic stem cells can be stimulated, make hematopoietic stem cell showed increased；Significantly improve intracellular mitochondrial number Mesh；Damage to liver, spleen has obvious restitution.This alimentation composition is applied to malnutritional anemia, and (iron-deficient is lean Blood and Folic Acid and vitamin b12Malnutritional anemia), erythropoiesis reduce Anemia and hematoclasis excessive or Lose Anemia.The Chinese invention patent of Patent No. 201010129030.4 also discloses this alimentation composition and can promote liver Dirty stem cells hyperplasia.I.e. alimentation composition only can promote the propagation of hematopoietic stem cell and liver stem cells it is impossible to meet other The needs of stem cells hyperplasia.
Content of the invention
The present invention is to solve the above-mentioned technical problem existing for prior art, providing one kind to have promotion Hematopoietic Stem thin The alimentation composition of five kinds of stem cells hyperplasias such as born of the same parents, mescenchymal stem cell and the application in preparation stem cells hyperplasia medicine.
The technical solution of the present invention is: a kind of alimentation composition is it is characterised in that each constituent mass ratio is as follows: bad ammonia Acid 200 ~ 1200, methionine 100 ~ 1600, Phenylalanine 150 ~ 1400, threonine 50 ~ 600, tryptophan 50 ~ 400, arginine 200 ~ 1500, histidine 100 ~ 1000, glycine 100 ~ 600, aspartic acid 100 ~ 600, leucine 100 ~ 600, isoleucine 100 ~ 600, L-Valine 100 ~ 800, serine 100 ~ 400, L-Glutamine 200 ~ 600, taurine 100 ~ 500, orotic acid 100 ~ 300th, nucleotide 200 ~ 1200, vitamin a 0.2 ~ 0.6, vitamin d 0.002 ~ 0.006, vitamin b21 ~ 4, vitamin b61 ~ 4, vitamin b120.001 ~ 0.006, nicotinic acid 5 ~ 20, Folic Acid 0.01 ~ 1.2, vitamin c 50 ~ 150, ferrum 2 ~ 10, zinc 2 ~ 10, Manganese 2 ~ 10, copper 0.5 ~ 2, selenium 0.01 ~ 0.1, chromium 0.01 ~ 0.02, potassium 10 ~ 300, calcium 100 ~ 300, magnesium 100 ~ 200, inositol 50 ~ 60th, soybean phospholipid 100 ~ 2500.
Each component optimum quality ratio is as follows: lysine 600, methionine 800, Phenylalanine 700, threonine 300, color ammonia Acid 200, arginine 760, histidine 500, glycine 300, aspartic acid 300, leucine 300, isoleucine 300, L-Valine 400th, serine 200, L-Glutamine 300, taurine 500, orotic acid 300, nucleotide 400, vitamin a 0.6, vitamin D0.006, vitamin b22nd, vitamin b62nd, vitamin b120.004th, nicotinic acid 12, Folic Acid 0.9, vitamin c 100, ferrum 10, zinc 10, Manganese 8, copper 2, selenium 0.1, chromium 0.02, potassium 300, calcium 300, magnesium 200, inositol 60, soybean phospholipid 1200.
Upper described alimentation composition promotes the application in stem cells hyperplasia medicine in preparation.
Upper described alimentation composition promotes the application in hemopoietic stem cell proliferation medicine in preparation.
Upper described alimentation composition promotes the application in proliferation of liver stem cells medicine in preparation.
Upper described alimentation composition promotes the application in mescenchymal stem cell hyperproliferation agent in preparation.
Upper described alimentation composition promotes the application in kidney stem cells hyperplasia medicine in preparation.
Upper described alimentation composition promotes the application in gastric mucosa stem cells hyperplasia medicine in preparation.
The alimentation composition of the present invention has promotion hematopoietic stem cell, liver stem cells, mescenchymal stem cell, kidney do thin Born of the same parents' propagation and the effect of gastric mucosa stem cells hyperplasia, can be applicable to preparation and promote stem cells hyperplasia medicine, nontoxic, have no side effect, Avoid giving exogenous stem cells carrying out the various problems existing for disease treatment.
Brief description
Fig. 1 is the impact schematic diagram to aplastic anemia rat body weight for the embodiment of the present invention.
Fig. 2 is the impact schematic diagram to aplastic anemia rat marrow hematopoietic stem cell for the embodiment of the present invention.
Fig. 3 is the impact schematic diagram to aplastic anemia rat liver stem cell for the embodiment of the present invention.
Fig. 4 is the impact schematic diagram to aplastic anemia rat kidney stem cell for the embodiment of the present invention.
Fig. 5 is the impact schematic diagram to aplastic anemia rat stomach stem cell for the embodiment of the present invention.
Fig. 6 is the impact schematic diagram to aplastic anemia Intestinal Mucosa stem cell for the embodiment of the present invention.
Fig. 7 is the impact schematic diagram to aplastic anemia neural stem cells in rats for the embodiment of the present invention.
Fig. 8 is the impact schematic diagram to aplastic anemia pancreas in rat stem cell for the embodiment of the present invention.
Fig. 9 is the impact schematic diagram to aplastic anemia rat muscle stem cell for the embodiment of the present invention.
Figure 10 is the impact schematic diagram to aplastic anemia rat bone marrow mesenchymal stem cellses for the embodiment of the present invention.
Specific embodiment
Embodiment 1: alimentation composition each constituent mass (mg) ratio of the present invention is as follows: lysine 200 ~ 1200, first sulfur ammonia Acid 100 ~ 1600, Phenylalanine 150 ~ 1400, threonine 50 ~ 600, tryptophan 50 ~ 400, arginase 12 00 ~ 1500, histidine 100 ~ 1000, glycine 100 ~ 600, aspartic acid 100 ~ 600, leucine 100 ~ 600, isoleucine 100 ~ 600, L-Valine 100 ~ 800, serine 100 ~ 400, L-Glutamine 200 ~ 600, taurine 100 ~ 500, orotic acid 100 ~ 300, nucleotide 200 ~ 1200th, vitamin a 0.2 ~ 0.6, vitamin d 0.002 ~ 0.006, vitamin b21 ~ 4, vitamin b61 ~ 4, vitamin b12 0.001 ~ 0.006, nicotinic acid 5 ~ 20, Folic Acid 0.01 ~ 1.2, vitamin c 50 ~ 150, ferrum 2 ~ 10, zinc 2 ~ 10, manganese 2 ~ 10, copper 0.5 ~ 2, selenium 0.01 ~ 0.1, chromium 0.01 ~ 0.02, potassium 10 ~ 300, calcium 100 ~ 300, magnesium 100 ~ 200, inositol 50 ~ 60, soybean phospholipid 100 ~ 2500.
Embodiment 2: each constituent mass (mg) ratio is as follows: lysine 600, methionine 800, Phenylalanine 700, threonine 300th, tryptophan 200, arginine 760, histidine 500, glycine 300, aspartic acid 300, leucine 300, isoleucine 300th, L-Valine 400, serine 200, L-Glutamine 300, taurine 500, orotic acid 300, nucleotide 400, vitamin a 0.6th, vitamin d0.006, vitamin b22nd, vitamin b62nd, vitamin b120.004th, nicotinic acid 12, Folic Acid 0.9, vitamin c 100, Ferrum 10, zinc 10, manganese 8, copper 2, selenium 0.1, chromium 0.02, potassium 300, calcium 300, magnesium 200, inositol 60, soybean phospholipid 1200.
Described alimentation composition has promotion hematopoietic stem cell, liver stem cells, mescenchymal stem cell, the increasing of kidney stem cell Grow and gastric mucosa stem cells hyperplasia effect, can be applicable to preparation promote stem cells hyperplasia medicine.
Embodiment 1,2 raw material sources are as follows:
Experiment:
One. the foundation of aplastic anemia rat model
This experiment adopts sd rat.Experimental procedure: after x-ray 2.5gy was irradiated in first day, gave ring phosphorus respectively in the 4th, 6,8 days Amide 35mg/kg, chloromycetin 45 mg/kg lumbar injection.15th day repeats above step.Corresponding with simple normal saline Injection location is Normal group.
Experiment packet: the drug dose according to animal pharmacology and the design analysis of compbined test, it is divided into: 1. normal control Group；2. model with aplastic anemia group；3. embodiment 2 alimentation composition high dose group, with 2266.95mg/kg.d to rat oral gavage；4. implement Example 2 alimentation composition middle dose group, with 1511.3mg/kg.d to rat oral gavage；5. embodiment 2 alimentation composition low dose group, With 1057.91mg/kg.d to rat oral gavage.
Experiment process: first, set up model by the method for building up of aforementioned aplastic anemia rat model, from the beginning of the 5th day, nutrition group The high, medium and low dosage group of compound carries out gavage with corresponding dosage to rat respectively, once a day, until the 60th day, draw neck to put to death big Mus, sampling is detected.Carry out whole observation simultaneously daily, measure body weight.
Two. experimental technique
1. peripheral blood detection
Each experimental group the 60th day, animal of weighing, take blood through eye socket, peripheral blood is measured using Automatic Blood Cell Analyzer blood red Protein content, erythrocyte, leukocyte and platelet count；Basis of microscopic observation peripheral blood film.
2. erythropoietin (epo) detection
ELISA Plate every hole addition titer, comparison liquid and the testing sample 100ul being diluted with sample diluting liquid in advance, shrouding, 37 ° C is incubated 90 minutes.After drying in the hole liquid after reaction, ready biotin anti-mouse epo antibody working solution is pressed every hole 100ul sequentially adds (except tmb blank colour developing hole), and after shrouding, 37 ° of c react 60 minutes, and 0.01m pbs washs 3 times, soak every time Bubble 1 minute about.Ready Avidin-peroxydase complex working solution is added to sequentially add (tmb by every hole 100ul Except blank colour developing hole), 37 ° of c react 30 minutes.Sequentially add balance tmb colour developing in 30 minutes in 37 DEG C by every hole 90 l Liquid, 37 ° of c lucifuge reactions, every hole 100 l sequentially adds tmb terminate liquid.Read absorbance (od with microplate reader at 450nm wavelength Value), sample epo concentration (pg/ml) is recorded according to standard curve.
3. bone marrow detection
Cervical dislocation put to death rat, separate bilateral femur open medullary cavity carry out bone marrow smear, BMNC count, The analysis of marrow protection inspection, Proliferation of Bone Mesenchymal Stem Cells and differentiation due.
4. the separation and Extraction of mesenchymal stem cells MSCs (bmscs)
After sd rats by intraperitoneal injection anesthetics, de- neck is put to death, and 75% ethanol soaks rat 20 min；Aseptic take double hind legs, put into 5 min are soaked in 75% ethanol；Remove lower limb muscles, from knee joint dialysis femur, cut off femur two ends；Injected with 10 ml The f-12k culture fluid that 10% hyclone drawn by device rinses medullary cavity, collects bone marrow irrigation liquid, makes single cell suspension, 200 mesh With 1 × 10 after screen cloth filtration8It is inoculated in culture bottle.It is placed in 37 ° of c, cultivate in 5% co2 incubator.Change first within 72 hours Culture fluid, removes non-attached cell, changes liquid 1 time every 2 days later.When cell is paved with the 80% ~ 90% of whole culture bottle floor space When, Secondary Culture.
5. the adipogenic induction differentiation of mescenchymal stem cell (bmscs)
Adipogenic induction method: by the mesenchymal stem cells MSCs isolating and purifying cellar culture, had digestive transfer culture, with 1 × 105/ hole close Degree, 400ul/ hole is inoculated in 6 well culture plates being pre-placed coverslip, prepares cell climbing sheet, when cell growth reaches 80% During fusion, the hyclone of addition 10%, 1 μm of ol/l dexamethasone, 0.5 mmol/l ibmx, h- of 10 mg/l bovine insulins Dmem induces 3 days, then the h-dmem of the hyclone with 10%, 10 mg/l bovine insulins is processed 1 day, after so circulating 3 times, Processed 7 days with the h-dmem of 10% hyclone, 10 mg/l bovine insulins, changed liquid 1 time every three or four days.Matched group adds all the time Enter containing volume fraction be 10% hyclone, 10 mg/l bovine insulins h-dmem, changed liquid 1 time every three or four days.After induction Cell pbs washs 2 ~ 3 times, and 10% formaldehyde room temperature fixes 40min.Pbs washes 2 ~ 3 times, and saturation oil red o dye liquor steams water with 3 with one: 2 dilutions, room temperature dyes 30min, and pbs washes for several times until being no observed visually sediment, om observation.
6. mescenchymal stem cell (bmscs) osteoblasts cultivation and identification
Take p2 for mesenchymal stem cells MSCs, by 2 × 105Individual/hole is inoculated in 12 orifice plates, when cell attachment growth reaches 80% fusion When, absorb culture fluid in hole, be changed to osteoblast induction liquid (containing high sugar dmem, volume fraction be 10% hyclone, 10-8 Mol/l dexamethasone, 10-2Mol/l sodium β-glycerophosphate, 50 mg/l ascorbic acid), every 3 days replacing induction liquid 1 time.Culture After 14 days, absorb culture fluid, pbs washes 2 times, and 10% formaldehyde room temperature fixes 40 min, and pbs washes 3 times, add 0.1% alizarin red agent (3:2 Dilution), room temperature dyes 10min, removes dye liquor, after pbs washes 3 times, basis of microscopic observation.
7. rna extracts and rt-pcr analysis
Conventional application trizol method extracts total rna of each specimen, and ultraviolet spectrophotometer measures a260And a280, to detect that rna contains Amount and purity, and it is placed in -70 ° of c preservations.
Reverse transcription system 20 μ l, comprises sample rna 1 μ l, the description that the reverse transcription reagent box according to takara provides Operated.Reverse transcription reaction condition is: 65 ° of c 1min, 30 ° of c 5min, 65 ° of c 15min, 98 ° of c 5min, 5 ° of c 5min.
Pcr reaction system 50 μ l, comprises reverse transcription liquid 10 μ l.Pcr reaction condition: 94 ° of c denaturations 1min；97°c Degeneration 20s, 64 ° of c annealing 20s, 72 ° of c 20s extend, and circulate 30 times；72 ° of c extend 5min, and 4 ° of c are constant.Take 5 μ l pcr Amplified production through 1% agarose gel electrophoresiies, gel imaging under uviol lamp.
8. transmission electron microscope observing
3 sections taking different parts in rat portions liver, spleen, brain, nephridial tissue specimen are carried out transmission electron microscope (jem-100cxe Type) Ultrastructural observation, every section respectively random observation 5-6 position, and take the photograph piece by identical amplification, every group with Machine extracts l0 and opens photo, using epson microcomputer and summa sketch pius digitizer, and uses sigmascan software, The form making sem image to each group photo is analyzed, and counts mitochondrial average number simultaneously.
9. mitochondrial membrane potential measures
Cell (5 × 105Individual) it is inoculated in 25cm2In culture bottle, after 37 DEG C of culture 24 h, the curcumin of variable concentrations is added to divide After not acting on 1 h and 6 h, harvesting, pbs washes twice, is resuspended in the fresh cultured that 2ml contains 1.0 m Rhodamine 123s In liquid, 37 ° of c water-bath shaking incubation 10 min, cell is removed in centrifugation, and in Fluorescence Colorimeter mensure culture fluid, Rhodamine 123 is strong Degree, excitation wavelength 490 nm, launch wavelength 520 nm, the fluorescence intensity of the Rhodamine 123 that result is absorbed with cell represents.
10. mitochondrial dna assay
By BMNC (5 × 107), flesh tissue liver, spleen, brain, kidney homogenate, cracking, centrifugation, obtain mitochondrion. Illustrate to extract the dna in mitochondrion according to dna extracts kit, its content is measured using ultraviolet spectrophotometer.Computing formula As follows:
Dna concentration (μ g/ μ l)=a260× 50 μ g/ml × extension rate × 10-3
11. pathological observations
Rat portions liver, spleen, brain, kidney, intestinal, muscle are placed in 4 % paraformaldehydes and fix, sequentially passes through conventional dehydration, paraffin Soak embedding, section, he dyeing, micro- sem observation each group knits the morphosiss of cell.
12. immunohistochemical stainings
Paraffin section de-waxing, aquation；Pbs wash 2-3 time each 5 minutes；3% h2o2On slide, room temperature stands (80% methanol) Deca 10 minutes；Pbs wash 2-3 time each 5 minutes；Antigen retrieval；Pbs wash 2-3 time each 5 minutes；Deca Normal Goat Serum confining liquid, room Temperature 20 minutes.Get rid of surplus liquid, Deca one resists 50 μ l, and 4 ° of c are overnight.Overnight afterwards need to be in 37 ° of c rewarmings 45 minutes.Pbs washes 3 times Each 5 minutes；The anti-40-50 μ l of Deca two, room temperature stands, or 37 ° of c 1 hour；Pbs wash 3 times each 5 minutes；Dab colour developing 5-10 divides Clock, grasps dye levels under the microscope；Pbs or tap water rinse 10 minutes；Haematoxylin redyeing 2 minutes, hydrochloride alcohol breaks up； Tap water rinses 10-15 minute；Dehydration, transparent, mounting, microscopy.
13. flow cytometry analysis
Take rat left tibia, remove superficial musculature, prune bone dirt, insert tibia ankle end with No. 18 pins, with pbs 5ml punching Wash in test tube, with 200 mesh sieve net filtrations.1000 r/min centrifugation 5min, abandon supernatant.Cell is added 5ml Percoll separating liquid (proportion 1.073g/l relatively), 2000r/min, it is centrifuged 25min, take middle mononuclearcell layer.Pbs washes After washing, adjustment mononuclearcell number is l × 106/ pipe, every sample 3 is managed, and measures cd34 the or cd45 antibody that pipe adds fitc labelling, 37 ° of c are incubated 2 hours.Then pbs washed once.Changed with flow cytomery expression cd34/45 positive cell quantity, with The cell percentage that traget antibody is positive is as the metering mark of expression cd34/45 albumen (marrow hemopoietic stem cells mark) Accurate.Negative control is made with the cell being not added with antibody with the goat-anti rabbit igg of fluorescein of only labelling simultaneously.
14. protein chip analyses
Rat portions BMNC, mesenchymal stem cells MSCs, liver organization are carried out protein chip analysis, this part Work is completed by upper Cannes Garden company.
15. statistical analysis
All data all carry out statistical analysiss with spss 17.0 software.At least three times results of each analysis.Numerical value mean ± sd represents, using t inspection.P < 0.05 thinks there is significant difference, and statistically meaningful.
3rd, experimental result
(1) impact to aplastic anemia rat body situation for the alimentation composition
Compared with Normal group, model with aplastic anemia group rat occurred successively from 6 days fur relax fluffy and disorderly, glossiness reduce, portion Rat is divided to have depilation and skin lesion.Rat becomes thin simultaneously, spirit is faded in not good, color of the lip eyelid is pale, movable minimizing, few food, body weight Decline.Wherein model with aplastic anemia group rat body weight (218.23 ± 33.52 g) and Normal group (366.54 ± 49.68 g, *p< 0.05) compare decline substantially.
The alimentation composition group of various dose is compared with model with aplastic anemia group, and the rat mental status is gradually good, and food-intake increases, body Increase (see figure 1) again.Show that alimentation composition has notable restitution to Induced Aplastic Anemia Mice whole body.
(2) impact to aplastic anemia rat peripheral blood for the alimentation composition
Compared with Normal group, erythrocyte (rbc), leukocyte (wbc), platelet (plt) in model with aplastic anemia group peripheral blood And hemoglobin (hb) is all remarkably decreased (table 1, #p < 0.05)；Various dose alimentation composition group and model with aplastic anemia group ratio Relatively, in its peripheral blood, indices all significantly raise (table 1, * p < 0.05), and are in dose-effect dependency.
Compared with Normal group, model with aplastic anemia group EPO of rats (epo) content is significantly raised, and epo Reduction with rbc, hb is in negative correlation, r=-0.91 and r=-0.93(*p< 0.05), show epo level and the red system of aplastic anemia bone marrow Generative capacity reduces, epo reactivity reduced capability, and epo is in that compensatory rising is relevant.(table 1, *p<0.01).Epo is a kind of weight The hematopoietic regulation factor wanted, has promotion CFU-E propagation, differentiation and ripe, erythrocyte in maintenance peripheral blood, blood red egg The effect of Bai Hengding, in blood, epo level can reflect the erythropoiesis ability in body.And the alimentation composition of various dose Group is compared with model with aplastic anemia group, and erythropoietin (epo) reduces (* due to compensation contentp<0.01)；And be in agent Amount-effect relation.
Result above is pointed out, and to aplastic anemia rat peripheral blood, various hemocytees have promotion rising effect to alimentation composition.
The impact to aplastic anemia rat peripheral blood for table 1 alimentation composition
#P < 0.05 aplastic anemia group and normal group；*P < 0.05 alimentation composition group and aplastic anemia group
(3) impact to aplastic anemia Murine Stem for the embodiment of the present invention alimentation composition
1. marrow hemopoietic stem cells
It is expressed on hematopoietic stem cell film to cd34 selection of antigen, be the mark of hematopoietic stem cell.Cd45 is single transmembrane sugar egg In vain, belong to protein tyrosine phosphatase family member, be widely present in hemopoietic stem cell surface.This experiment adopts SABC to examine Survey the expression of aplastic anemia rat marrow hematopoietic stem cell mark cd34, cd45.Result is as shown in Fig. 2 and Normal group Compare, the expression of model with aplastic anemia group rat marrow hematopoietic stem cell cd34, cd45 significantly reduces, and shows that aplastic anemia rat marrow is made Hemocytoblast quantity reduces；After alimentation composition intervenes aplastic anemia rat, the expression of rat marrow hematopoietic stem cell cd34, cd45 It is in dose dependent with alimentation composition, show that alimentation composition has the work promoting aplastic anemia rat marrow hemopoietic stem cell proliferation With.
2. liver stem cells
Cd90 is a kind of cell surface glycoprotein, is the mark of liver stem cells, is also that the antigen of blood stem cell is determined simultaneously Determine cluster.Hepatic oval cells are a kind of more liver stem cells of current research, and its surface has specific marker cytokeratin 19(ck19) expression is higher.This experiment adopts SABC to detect the expression of aplastic anemia Rat Hepatic Stem Cells mark cd90, ck19 Situation.Result is as shown in figure 3, compared with Normal group, model with aplastic anemia group rat liver stem cell population significantly reduces, nutrition After compositions-treated aplastic anemia rat, the content of rat liver stem cell increases with alimentation composition dosage and increases, and shows nutrition Compositionss can promote the propagation of aplastic anemia rat liver stem cell.
3. kidney stem cell
In kidney, cd133 is distributed in renal medulla mamillary region, and ability of cell proliferation is strong it is believed that this area is kidney stem cell settlement. Oct4 is the transcription factor maintaining stem cell versatility and self renewal.This experiment adopts SABC to detect aplastic anemia rat kidney The expression of stem cell markers cd133, oct4, result is as shown in figure 4, compared with Normal group, model with aplastic anemia group is big The expression of Mus cd133, oct4 significantly reduces, and after alimentation composition processes aplastic anemia rat, its expression is with nutrient combination agent Amount increases and increases, and shows that alimentation composition promotes the propagation of aplastic anemia rat kidney stem cell.
4. stomach stem cell
Lgr5 also known as gpr49, Recent study shows, lgr5 is the tumor stem cell labellings such as colorectal cancer, glue blastoma, all Label cd133, cd44 of relatively the past have more specificity.Musashi-1 mainly expresses in gastric epithelial isthmus, can be used as people The label of class normal gastrointestinal tract epithelial stem cell.This experiment adopts SABC to detect aplastic anemia rat stomach stem cell markers The expression of lgr5.Result is as shown in figure 5, compared with Normal group, model with aplastic anemia group rat stomach stem cell population significantly reduces； After alimentation composition processes aplastic anemia rat, the expression of rat stomach stem cell lgr5 increases with alimentation composition dosage and increases, table Bright alimentation composition has the propagation promoting aplastic anemia rat stomach stem cell.But each group stomach stem cell musashi-1's is no obvious Change.
5. intestinal mucosa stem cell
Little intestinal stem cell is a kind of undifferentiated initial cell, positioned at the nearly basilar parts of crypts of small intestine.There is self renewal and propagation It is divided into the function of various maturation intestinal epithelial cells.Research shows, musashi-1 is the selected marker of intestinal stem cell. Lgr5 limitation expression in the small intestinal and big intestinal crypt of muroid, is one of mark of intestinal stem cell.This experiment is using exempting from Epidemic disease groupization detects the expression of aplastic anemia intestine in rats stem cell markers musashi-1, lgr5.Result is as shown in fig. 6, each group is little The no significant change of intestinal stem cell quantity.Show the propagation no remarkable effect to aplastic anemia rat small intestine stem cell for the alimentation composition.
6. neural stem cell
Nestin is one of mark of neural stem cell or neural precursor during development of central nervous system.Cd133 is Hematopoietic stem cell, the mark of endothelial progenitor cells, later cd133 be proved that it, as a kind of stem cell labeling thing, is also current The brain Tumor Stem Cells relatively generally acknowledged and the surface marker of neural stem cell.This experiment adopts SABC to detect aplastic anemia rat The expression of neural stem cell mark nestin, cd133, result is as shown in fig. 7, alimentation composition processes aplastic anemia rat Afterwards, the expression of nestin, cd133 no significant changes.Show alimentation composition to the propagation of aplastic anemia rat stem cell of cranial nerve no Appreciable impact.
7. pancreatic stem cells
Cyfra21-1 (ck19) is the distinctive mark of epithelial cell, has positive expression in pancreatic stem cells.
Nestin is in the specific expression of neuroepithelial stem cell, and studies display at present, and nestin is in pancreatic stem cells In be also in high expression.This experiment adopts SABC to detect the expression of aplastic anemia pancreas in rat stem cell markers ck19, nestin Situation, result is as shown in figure 8, each group pancreatic stem cells quantity no significant change.Show alimentation composition to aplastic anemia pancreas in rat The propagation of stem cell no remarkable effect.
8. muscle stem cell
At present, flesh stem cell still lacks Specific marker.Desmin is considered in close relations with the early differentiation of myocyte, In Muscle-derived Stem Cells, the positive rate of desmin is up to 90%, frequently as the mark of flesh stem cell.Cd34 is the saliva of cell surface Liquid mucin is it is considered to be the label of hematopoietic stem cell, but also has obvious expression in flesh stem cell.This experiment is using immunity Groupization detects the expression of aplastic anemia rat muscle stem cell markers desmin, cd34.Result is as shown in figure 9, each group intestinal is done carefully The no significant change of born of the same parents' quantity.Show the propagation no obvious effect to aplastic anemia intestine in rats stem cell for the alimentation composition.
9. mesenchymal stem cells MSCs
Each group Proliferation of Bone Mesenchymal Stem Cells capability result is shown in Figure 10, and aplastic anemia group is substantially less than normal group, low dose group with again Barrier group zero difference, middle dose group, high dose group and aplastic anemia group have notable difference.Show alimentation composition between aplastic anemia rat marrow The propagation of mesenchymal stem cells has obvious effect.

Claims (8)

1. a kind of alimentation composition is it is characterised in that each constituent mass ratio is as follows: lysine 200 ~ 1200, methionine 100 ~ 1600th, Phenylalanine 150 ~ 1400, threonine 50 ~ 600, tryptophan 50 ~ 400, arginase 12 00 ~ 1500, histidine 100 ~ 1000th, glycine 100 ~ 600, aspartic acid 100 ~ 600, leucine 100 ~ 600, isoleucine 100 ~ 600, L-Valine 100 ~ 800th, serine 100 ~ 400, L-Glutamine 200 ~ 600, taurine 100 ~ 500, orotic acid 100 ~ 300, nucleotide 200 ~ 1200, Vitamin a 0.2 ~ 0.6, vitamin d 0.002 ~ 0.006, vitamin b21 ~ 4, vitamin b61 ~ 4, vitamin b120.001~ 0.006th, nicotinic acid 5 ~ 20, Folic Acid 0.01 ~ 1.2, vitamin c 50 ~ 150, ferrum 2 ~ 10, zinc 2 ~ 10, manganese 2 ~ 10, copper 0.5 ~ 2, selenium 0.01 ~ 0.1, chromium 0.01 ~ 0.02, potassium 10 ~ 300, calcium 100 ~ 300, magnesium 100 ~ 200, inositol 50 ~ 60, soybean phospholipid 100 ~ 2500.
2. alimentation composition according to claim 1 is it is characterised in that each constituent mass ratio is as follows: lysine 600, first sulfur Propylhomoserin 800, Phenylalanine 700, threonine 300, tryptophan 200, arginine 760, histidine 500, glycine 300, Radix Asparagi ammonia Acid 300, leucine 300, isoleucine 300, L-Valine 400, serine 200, L-Glutamine 300, taurine 500, orotic acid 300th, nucleotide 400, vitamin a 0.6, vitamin d0.006, vitamin b22nd, vitamin b62nd, vitamin b120.004th, nicotinic acid 12nd, Folic Acid 0.9, vitamin c 100, ferrum 10, zinc 10, manganese 8, copper 2, selenium 0.1, chromium 0.02, potassium 300, calcium 300, magnesium 200, flesh Alcohol 60, soybean phospholipid 1200.
3. a kind of alimentation composition as claimed in claim 1 or 2 promotes the application in stem cells hyperplasia medicine in preparation.
4. according to claim 3 alimentation composition preparation promote stem cells hyperplasia medicine in application it is characterised in that Promote the application in hemopoietic stem cell proliferation medicine in preparation.
5. according to claim 3 alimentation composition preparation promote stem cells hyperplasia medicine in application it is characterised in that Promote the application in proliferation of liver stem cells medicine in preparation.
6. according to claim 3 alimentation composition preparation promote stem cells hyperplasia medicine in application it is characterised in that Promote the application in mescenchymal stem cell hyperproliferation agent in preparation.
7. according to claim 3 alimentation composition preparation promote stem cells hyperplasia medicine in application it is characterised in that Promote the application in kidney stem cells hyperplasia medicine in preparation.
8. according to claim 3 alimentation composition preparation promote stem cells hyperplasia medicine in application it is characterised in that Promote the application in gastric mucosa stem cells hyperplasia medicine in preparation.
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