CN106692951A - Composition and application thereof - Google Patents

Composition and application thereof Download PDF

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Publication number
CN106692951A
CN106692951A CN201611220196.0A CN201611220196A CN106692951A CN 106692951 A CN106692951 A CN 106692951A CN 201611220196 A CN201611220196 A CN 201611220196A CN 106692951 A CN106692951 A CN 106692951A
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China
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composition
cell
liver
group
mesenchymal stem
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CN201611220196.0A
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Chinese (zh)
Inventor
葛啸虎
陈海佳
王飞
王一飞
黄新珠
王小燕
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Guangzhou Saliai StemCell Science and Technology Co Ltd
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Guangzhou Saliai StemCell Science and Technology Co Ltd
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Priority to CN201611220196.0A priority Critical patent/CN106692951A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • 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
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins

Abstract

The invention relates to the field of cells, and in particular to a composition and an application thereof. The composition consists of a growth factor, albumin and mesenchymal stem cells. The composition is effective in preventing and/or treating chronic liver injuries caused by ischemia, drugs (such as paracetamol), viruses, obesity, non-alcoholic steatohepatitis, biliary obstruction and tumors. The composition can also prevent hepatic failure, retard the progress of chronic hepatic failure and remove harmful toxin.

Description

Composition and its application
Technical field
The present invention relates to cell field, more particularly to composition and its application.
Background technology
Hepatic failure is that the major Liver that many factors cause is damaged, and causes the work(such as its synthesis, removing toxic substances, excretion and bioconversion Serious hindrance or decompensation can occur, it is the one of main performance to occur with coagulation function obstacle and yellow subcutaneous ulcer, hepatic encephalopathy, ascites etc. Group clinical syndrome.In the last few years, because the dead number of hepatic failure gradually increases, according to statistics, it is dead that the U.S. there are about ten thousand people every year In liver failure, and China dies from the patient Yue Wan of this disease every year.Acute hepatic failure is a kind of acute serious liver function energy barrier Hinder, be characterized in that onset is anxious, the state of an illness is critical, Symptoms are various, liver cell extensive necrosis.The primary hand of hepatic failure is treated at present Section is medical treatment, liver transfer operation and cell therapy etc..Medical treatment offer limited effectiveness, not yet there is specific drug to the ill so far. At present, though liver transfer operation turned into treatment End-stage liver disease important means, donor shortage, surgical injury, immunological rejection and The problems such as high cost, constitutes the major obstacle of Technology of Liver Transplantation in China development.Therefore, carrying out has invasive small, few intercurrent disease etc. The stem cell transplantation of feature turns into the study hotspot that treatment liver declines recently.Stem cell is a kind of undifferentiated cell, there is two bases Eigen, one is the of self-replication capacity, and two is that can be divided into more than one functioning cell, according to the size of differentiation potential, is done Cell is broken generally into three classes, and the first kind is that myeloid-lymphoid stem cell can be categorized as the consistent totipotent cell of function.Equations of The Second Kind is many Pluripotent stem cell, it can break up as every kind of cell type in body, but necessary to can not forming placenta and development of fetus Supporting tissue.3rd class is that adult stem cell is a kind of stem cell that will possess lifelong self-replacation or self-renewal capacity.It It is all kinds cell to be distributed in different tissues and can form.
Mescenchymal stem cell, belongs to adult stem cell, be it is a kind of with the of self-replication capacity and multi-lineage potential into Somatic stem cell, belongs to non-terminally differentiated cells, can be divided into fat, cartilage, bone, muscle, flesh leg, nerve, liver, cardiac muscle, pancreas islet The Various Tissues cell such as cell and endothelium.Mescenchymal stem cell is low due to immunogenicity, and adult tissue's abundance is widely present Marrow, adipose tissue, neonatal umbilical cord and placenta in human body etc., it is easy to separate, purity is high, during negative for tumor cells pollution amplification Cultivating system can be unified, and be easy to Quality Control to can be made into seed cell and freeze, and be used for multiple times, the small latent virus of loss cell after freezing To puerpera and neonate without any harm and damage collection side during with the infection of pathogenic microorganism and propagation probability than relatively low collection Just, it is easy to storage and transport, ethics dispute is few.Many researchs at present have shown that stem cell entered specific culture and has point in vitro It is melted into the ability for liver cell.
The mescenchymal stem cell of derived from bone marrow in recent years, because it has, favorable reproducibility, low immunogenicity, expense be low, fortune for treatment Make the advantage such as convenient, easy, as the effective ways after another treatment hepatic failure after orthotopic liver transplantation.Preparation process is relatively simple It is single, be easy to collect, it is biological nature stabilization, security performance stabilization, reproducible, patient can psychologically receive, therefore can be completely The demand of sufficient clinical practice.Multiplication capacity is strong, and there is adherence quality to be conducive to the purifying and culture of cell for it, while having stronger Differentiation capability, immunogenicity are low, avoid embryo stem cell transplantation after there may be teratoma and ethics problem；It can be different Gene transplant long-term survival simultaneously plays normal physiological function, and autotransplantation does not exist immune rejection problems, therefore newly Treatment method one have great advantage in treatment.
Many researchs at present have shown that stem cell entered specific culture and has ability of the differentiation as liver cell in vitro.Prove Candidate stem cell be able to can be broken up as liver cell with vitro culture.Research has shown that the derived mesenchymal stem cells in vitro point of derived from bone marrow Changing culture can break up hepatoblast.The relevant experimental model of current rat, rat and people, points out bone marrow-derived cells to turn Turn to liver cell.The current report on separate sources stem-cell therapy acute hepatic failure also has a lot.Prove that single bony nodule marrow is thin Born of the same parents can treat the acute hepatic failure caused to second phthalidyl amino phenols.Someone draws a conclusion, and some partial injuries may be more suitable in liver The stem cell for sharing derived from bone marrow is repaired.
Hwang, S. et al. are filled using mesenchymal stem cells MSCs and through between the HGF marrow of external evoked 1 week or 2 weeks respectively Matter stem-cell therapy hepatic failure rat, by the detection to liver function blood biochemistry index, they think stem cell to liver stem cells The early stage broken up with liver cell is advantageously possible for the regeneration of liver cell and the reconstruction of liver function, and ripe hepatocyte transplantation may The recovery of liver function can be limited.
The content of the invention
In view of this, the present invention provides a kind of composition and its application.Said composition is for preventing and/or treating by part Ischemic, medicine such as paracetamol, virus, obesity nonalcoholic fatty liver disease and obstruction bile duct obstruction, tumour institute The chronic liver injury for causing is effective.Said composition can also prevent hepatic failure, slow down chronic liver failure is in progress and removes harmful Toxicity.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of composition, including growth factor, albumin and mesenchymal stem cells MSCs.
In some specific embodiments of the invention, the growth factor be selected from HGF, prostaglandin, Insulin or hyperglycemic factor.
In some specific embodiments of the invention, HGF accounts for the content of composition in the composition For the content that 30 μ g/ml~60 μ g/ml, prostaglandin account for composition is that 4 μ g/ml~8 μ g/ml, insulin account for containing for composition Measure as the content that 20U/ml~40U/ml, hyperglycemic factor account for composition is 3mg/ml~6mg/ml.
In some specific embodiments of the invention, albumin described in the composition accounts for the quality percentage of composition Content is 15~30%.
In some specific embodiments of the invention, albumin described in the composition accounts for the quality percentage of composition Content is 20%.
In some specific embodiments of the invention, the concentration of mesenchymal stem cells MSCs is described in the composition 1.0~10.0 × 107/ml。
In some specific embodiments of the invention, the concentration of mesenchymal stem cells MSCs is described in the composition 1.5×107/ml。
Liver regeneration Initiated Mechanism is complicated, is related to various kinds of cell, cell factor and growth factor.Wherein HGF It is to promote one of most important growth factor in Hepatopoietin and use thereof, is mainly produced by interstitial cells such as liver Kupffer cells, thin It is also signal necessary to liver regeneration starts with the effect for protecting liver cell, the apoptosis of suppression liver cell during cellular damage.Research Show, HGF can not only promote mature hepatocytes to breed, may also speed up propagation, the differentiation of hepatic oval cells, Liver regeneration is effectively facilitated in terms of mature hepatocytes and liver precursor two.
When liver cell is a large amount of downright bad, miopragia is inactivated to insulin, while the property serum insulin sample growth of liver cell source The factor 1 (insulin-like growth factor-1, IGF-1) expression is reduced, and hepatic failure patients hypoglycemia and sugar is occurred Oxidation utilization rate decline, turn based on protein, lipolysis energy supply.Hepatic failure patients are under stress situation using the energy of sugar Power is very poor, may not cause or aggravate the steatosis of liver by oxidation in time.Insulin and hyperglycemic factor combination not only may be used To improve the function that body is bred to the utilization rate of blood sugar and with cell cultured supernatant.
PGE2 (prostaglandin E2, PGE2) is arachidonic acid in phospholipase A2, epoxidase, and PGE2 Under the sequential catalyzed effect of synthase produce metabolite, can be synthesized by various kinds of cell, many reasons are caused including stomach and intestine Road mucous membrane, liver have protective effect in interior Various Tissues organ damage, when liver is subjected to Hepatoxic substance, it is possible to reduce Liver cell is adjusted and dies and destroy.
In some specific embodiments of the invention, growth factor described in the composition is selected from recombined human liver Growth factor, insulin, hyperglycemic factor or PGE2.
In some specific embodiments of the invention, the dosage of composition is between marrow described in the composition Mesenchymal stem cells are calculated as 1.0~10.0 × 107/ kg the weight of animals.
The medicine and/or health products of prevention and/or treatment liver diseases are being prepared present invention also offers the composition. The liver diseases are selected from by ischaemic, medicine such as paracetamol, virus, obesity nonalcoholic fatty liver disease, Chronic liver injury caused by obstruction bile duct obstruction, tumour, hepatic failure, or chronic liver failure.
Additionally, present invention also offers a kind of medicine and/or health products for preventing and/or treating liver diseases, its feature It is, including the composition that the present invention is provided.
The invention provides a kind of composition, including growth factor, albumin and mesenchymal stem cells MSCs.Said composition For prevent and/or treat by ischaemic, medicine for example paracetamol, virus, obesity nonalcoholic fatty liver disease, And obstruction bile duct obstruction, the chronic liver injury caused by tumour are effective.Said composition can also prevent hepatic failure, slow down slow Property hepatic failure is in progress and removes harmful toxicity.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing The accompanying drawing to be used needed for having technology description is briefly described.
Fig. 1 shows control group flow cytomery result；
Fig. 2 shows sample group flow cytomery result；
Fig. 3 shows that pathological section shows result；Wherein, Fig. 3 (A) shows normal group；Fig. 3 (B) shows control group；
Fig. 4 shows life cycle result.
Specific embodiment
The invention discloses a kind of composition and its application, those skilled in the art can use for reference present disclosure, suitably change Enter technological parameter realization.In particular, all similar replacements and change are for a person skilled in the art aobvious And be clear to, they are considered as being included in the present invention.The method of the present invention and application are carried out by preferred embodiment Description, related personnel can substantially be carried out not departing from present invention, spirit and scope to method described herein and application Change or suitably change with combining and realize and apply the technology of the present invention.
Raw materials used and reagent can be bought by market in the composition of present invention offer and its application.
With reference to embodiment, the present invention is expanded on further：
The mesenchymal stem cells MSCs of embodiment 1 is isolated and purified
Separate：
Short neck is put to death after giving rats by intraperitoneal injection heparin.Soaking disinfection 10min in 75% ethanol solution.Nothing in super-clean bench Rats with bilateral spinal joints is taken under the conditions of bacterium, is rinsed 2 times with PBS.The end of rat femur is cut off, anti-with syringe pump culture medium Ossis is rinsed again, and ossis is rinsed well.By cell suspending liquid removed by 70 μm of cell filtering net bone chip and The blood of aggregation.Cell suspension is collected with centrifuge tube, with culture medium cyclic washing 2 times, centrifugation.With the DMEM- containing 10%FBS F12 culture medium re-suspended cells, and cell density is adjusted for 2 × 105/ml is inoculated into 15cm culture dishes, it is transferred to 5%CO2,37 DEG C, cultivate in the CO2 incubators that saturated humidity is 95%.Half amount changes liquid during cell culture 24h, non-attached cell is removed, after 72h Secondary full dose changes liquid, and hereafter 3d changes liquid once, until cell growth to degrees of fusion up to 85%, pancreatin had digestive transfer culture or freezes.This It is Primary bone marrow mescenchymal stem cell.
Secondary Culture：After first all suctioning out nutrient solution during passage, wash secondary with PBS, add 37 DEG C the 0.25% of preheating (2.5g/L) trypsase (EDTA containing 1mmol/L is prepared with 1mmol/L-EDTA-Na2) is digested 2 minutes, and addition is trained completely Nutrient solution terminates pancreatin effect.Be collected in cell in 15ml centrifuge tubes after blowing and beating repeatedly by suction pipe, after 1500r/min is centrifuged 10 minutes Supernatant is abandoned, then is washed with PBS liquid and secondary is thoroughly washed away the trypsase being mixed with.Cell is resuspended in nutrient solution, by 2~5 × 105/ml is inoculated with passes in new blake bottle again.When these cell growths are close to fused layer, that is, obtain marrow MSC.
Mesenchymal stem cells MSCs amplification cultivation
Cell concentration is adjusted to 1 × 10 with L-DMEM complete culture solutions6/ mL cultivates mesenchymal stem cells MSCs cell. In 5%CO2 incubators, every 3~4 days half amounts change liquid 1 time, the cell factor needed for 2-3 days supplements；10~12d cells reach Passed on during 90% fusion, identify that mesenchymal stem cells MSCs and trypan blue count test cell vigor with flow cytometry.
The identification of mesenchymal stem cells MSCs：
Take the 3rd generation cell, cell culture to when fusion, 0.25% Trypsin Induced 2min, with containing 5%FBS Cold PBS rinse 2 times, add the PBS of 1ml 0.01mol/L resuspended, cell is gently blown and beaten repeatedly, make it into single cell suspension, CD29, CD90, CD11b, CD45 monoclonal antibody are added, while with Isotype control, lucifuge is incubated 1h, it is upper after PBS washes 3 times Formula is detected.Addition 1ml0.5% paraformaldehydes-PBS solution is fixed, re-suspended cell, flow cytometer detection.After isolating and purifying Rat peripheral blood NK cells through Trypan Blue, show that its activity can reach 92%~95%, through flow cytomery, CD73, CD90, CD105 ＞ 95%, CD19, CD34, CD11b, CD45 ＜ 2%, it was demonstrated that isolate and purify the medulla mesenchyma of amplification The purity of stem cells may be up to more than 95%.
Embodiment 2 builds rat chronic hepatic failure model
With intraperitoneal injection Carbon Tetrachloride Induced rat chronic hepatic failure model, carbon tetrachloride is according to 4:6 are dissolved in vegetable oil. Healthy week old adult male SD rats 60 are chosen, body weight (200 scholar 20) g is randomly divided into 4 groups：Normal group (25), model group (25), cell composition treatment group 1 (20), cell composition treatment group 2 (20), cell composition treatment group 3 (20 ) and physiological saline group (20).Model group, cell composition treatment group and physiological saline group are used and contain 40%CCl4Plant Thing oil solution 1ml/kg body weight carries out intraperitoneal injection, and every injection in three days once, week for 7 weeks induces model group rats Chronic Liver MSOF.Normal group carries out intraperitoneal injection using vegetable oil 1ml/kg body weight, every injection in three days once, week for 7 weeks.Model After making 7 weeks, randomly select normal group and model group rats each 5 only with automatic clinical chemistry analyzer detect serum and level with Observation mouse liver function damage situations, the pathological change of basis of microscopic observation mouse liver.After modeling, in model group mice serum AST and ALT water average specific normal groups are significantly raised；Pathological section shows, oedema occurs in model group murine liver tissue, vacuole, greatly Amount inflammatory cell infiltration, and have wall regeneration, be consistent with its morphology and biochemical indicator performance, then judge that Chronic Liver declines Exhaust modelling success.
Liver Function index serum ALB, ALT, AST and prothrombin time PT levels after the modeling of table 1
AST ALT ALB PT(s)
Normal group 30.46±15.35 175.62±23.75 43.52±6.44 17.86±5.21
Model group 276.79±38.70▲ 648.31±34.65▲ 16.71±2.67▲ 37.63±8.19▲
▲ P ＜ 0.05vs normal groups；
Rat blood serum ALT (ALT) and aspartate aminotransferase (AST) substantially rise after modeling Height, ALB is significantly lower than control group (P ＜ 0.05).
The preparation of the mesenchymal stem cells MSCs composition of embodiment 3
By flow cytometry identification and Trypan Blue, purity is more than 90% and sum reaches 107Marrow between fill Matter stem cell, centrifugation removal supernatant, plus injection physiological saline is cleaned 1-2 times, 400g centrifugation 5min, collects cell；
According to 30 μ g/ml HGFs, 4 μ g/ml prostaglandins, 20U/ml insulin, 3mg/ml pancreas hyperglycaemia Element, 20% (w/w) albumin, 1.5 × 107The mesenchymal stem cells MSCs of/kg rat body weights is resuspended in injection physiological saline In be made the mesenchymal stem cells MSCs composition of 1ml.
The preparation of the mesenchymal stem cells MSCs composition of embodiment 4
By flow cytometry identification and Trypan Blue, purity is more than 90% and sum reaches 107Marrow between fill Matter stem cell, centrifugation removal supernatant, plus injection physiological saline is cleaned 1-2 times, 400g centrifugation 5min, collects cell；
According to 40 μ g/ml HGFs, 6 μ g/ml prostaglandins, 30U/ml insulin, 5mg/ml pancreas hyperglycaemia Element, 15% (w/w) albumin, 10 × 107The mesenchymal stem cells MSCs of/kg rat body weights is resuspended in injection physiological saline It is made the mesenchymal stem cells MSCs composition of 1ml.
The preparation of the mesenchymal stem cells MSCs composition of embodiment 5
By flow cytometry identification and Trypan Blue, purity is more than 90% and sum reaches 107Marrow between fill Matter stem cell, centrifugation removal supernatant, plus injection physiological saline is cleaned 1-2 times, 400g centrifugation 5min, collects cell；
According to 60 μ g/ml HGFs, 8 μ g/ml prostaglandins, 40U/ml insulin, 6mg/ml pancreas hyperglycaemia Element, 30% (w/w) albumin, 1.0 × 107The mesenchymal stem cells MSCs of/kg rat body weights is resuspended in injection physiological saline In be made the mesenchymal stem cells MSCs composition of 1ml.
The tail vein of embodiment 6 is transplanted
Modeling carries out stem cell composition (3~embodiment of embodiment 5 is obtained) transplanting, after rat anesthesia, sterilization after 7 weeks Afterbody, gives injection in stem cell composition 1ml injection transplantations, 15min and finishes by tail vein.Saline control group and just Often group is used to same volume physiological saline tail vein.Once in a week, totally 8 weeks.
The main pharmacodynamics result of study of embodiment 7
After ordinary circumstance modeling, model group rats engender loss of appetite, and behavior is blunt and stimulation is quick on the draw The behaviouristics such as degree decline change.But compared with model group, by stem cell composition of the present invention treatment after, the dietary amount of rat and Behavior sensitivity increases, and above-mentioned symptom substantially mitigates, and the effect of stem cell composition of the present invention is more obvious.It is heavier Want, the adverse reaction related to stem cell is not found over the course for the treatment of.
Life cycle as shown in figure 4, analyzed by Kaplan-Meier, mean survival time of model group is 4.65 months, The mean survival time of growth factor control group is 5.15 months, the average life of stem cell group (embodiment 3~5 is obtained) of the present invention The time is deposited for 5.88 months, while being pointed out by inspection, the Cumulative survival rate of stem cell group of the present invention is higher than model group and growth Factor set, difference is statistically significant.
The influence (g/ days) of the rat diet amount of table 2
▲ P ＜ 0.05vs blank groups；△ P ＜ 0.05vs model groups；★ P ＜ 0.05vs growth factor groups；
Record rat diet, activity, to IR, fur gloss, depilation situations such as.Using comprehensive grading standard determination It is as follows:Movable normal (3 points of note) is 3~5 times/min, and it is 1~2 time/min that activity reduces (2 points of note), and inertia (1 point of note) is< 1 time/min.Be quick on the draw (remember 3 points) is to be rapidly converting to tap sound direction and with limb motion, reaction to beaing cage wall sound It is only to turn to percussion sound direction to beaing cage wall sound to weaken (2 points of note), and (1 point of note) slow in reacting is to tapping cage wall sound only Open eyes or come back.
As a result：
Normal rats are flexible, and reaction is sensitive, diet is normal, and fur light is submissive.Rat modeling after 1 month it is more normal Group and sham-operation group are slightly worse, and postoperative 21d appearance activity is reduced, and IR weakens to external world, and burnout is One's spirits are drooping, postoperative 28d Then above-mentioned performance is aggravated, and each basic inertia of rat is slow in reacting, and fur is confused and disordered, come off, easily concurrent various infection diseases. Each group activities in rats and reaction scoring situation are shown in Table 3.
The activities in rats of table 3 and reaction comprehensive grading compare
▲ P ＜ 0.05vs blank groups；△ P ＜ 0.05vs model groups；★ P ＜ 0.05vs growth factor groups；
Liver function and coagulation function
Treatment end each group rat adopts tail vein, and serum alt, AST, ALB water are detected with automatic clinical chemistry analyzer It is flat, detect liver function and coagulation function.
Normal group：Normal group carries out tail vein injection using physiological saline 1ml；
Model group：Model group carries out tail vein injection using physiological saline 1ml；
Growth factor control group：The same model group of modeling method, with containing 20% (v/v) albumin, 150 μ after modeling 7 weeks G/kg HGFs, 20 μ g/kg PGE2s, 0.1U/kg insulin, the physiology salt of 15 μ g/kg hyperglycemic factors Water 1ml carries out tail vein injection；
1~treatment group for the treatment of group 3 (stem cell group)：The same model group of modeling method, reality is given after 7 weeks in modeling to tail vein Applying composition 1ml obtained in example 3~5 carries out tail vein injection.
Liver biochemical indexes observation (x ± s) after the chronic liver failure mouse adipose mesenchymal stem cell transplantation of table 4
▲ P ＜ 0.05vs blank groups；△ P ＜ 0.05vs model groups；★ P ＜ 0.05vs growth factor groups；
As shown in table 4, after intraperitoneal injection CCl4 week for 7 weeks, model group rats Serum ALT, AST, TBIL and PT level show Write and raise, while serum ALB, CHE level is remarkably decreased, compare with blank group, difference is statistically significant.And pass through this hair After bright stem-cell therapy, Serum ALT, AST, TBIL and PT level are remarkably decreased, while serum ALB, CHE level significantly rises, Compared with model group and physiological saline group, difference is statistically significant.
Hepatic tissue pathology scores
Normal group：Normal group carries out tail vein injection using physiological saline 1ml；
Model group：Model group carries out tail vein injection using physiological saline 1ml；
Growth factor control group：The same model group of modeling method, with containing 20% (v/v) albumin, 150 μ after modeling 7 weeks G/kg HGFs, 20 μ g/kg PGE2s, 0.1U/kg insulin, the physiology salt of 15 μ g/kg hyperglycemic factors Water 1ml carries out tail vein injection；
1~treatment group for the treatment of group 3 (stem cell group)：The same model group of modeling method, reality is given after 7 weeks in modeling to tail vein Applying composition 1ml obtained in example 3~5 carries out tail vein injection.Method：
Materials → dehydration → waxdip → embedding → section → dyeing → sealing
Materials：At rat after death, respectively at the same position materials of liver, liver organization is cut into size with thin blade and is about The tissue block of 10mm × 10mm × 2mm, is placed in 10% neutral formalin solution and soaks fixed 48h；According to following procedure after fixation Carry out microsection manufacture：
Dehydration
Fixing organization blocks are immersed in the dehydrating agent of increasing concen-trations successively：75% ethanol soaks 2 hours, the leaching of 85% ethanol Bubble 2 hours, 95% ethanol soaks 1 hour, and 95% ethanol soaks 1 hour, and 100% ethanol I soaks 1 hour, 100% ethanol II leachings Bubble 1 hour；
It is transparent
Dimethylbenzene I soaks 1 hour, and dimethylbenzene II soaks 1 hour
Waxdip
Soaked in the paraffin of different melting points：Immersion 1 hour, paraffin II (soft waxs (54 in paraffin I (soft wax (52 DEG C)) DEG C)) soak 1 hour, paraffin III (hard wax (58 DEG C)) soaks 1 hour；
Embedding
Tissue block is pressed from both sides out with tweezers to be placed in container, add paraffin III, be subsequently placed into -20 DEG C of refrigerators, it is condensed into wax Block；
Section
The wax stone fixed and fixed is fixed on slicer folder thing platform, cuts out the section of some 5 μ m thicks, the wax for cutting Piece is placed in 40 DEG C of water-baths fully unfolds, and wax disk(-sc) is attached on slide, is dried overnight；
Dewaxing
Paraffin section is by dimethylbenzene II, dimethylbenzene I, 100% ethanol II, 100% ethanol I, 95% ethanol, 95% second Alcohol, 85% ethanol, 75% ethanol, each immersion 10min in distilled water；
Dyeing
Haematoxylin dyeing 5min, running water is rinsed.(blotting water)
Acidic alcohol differentiation 30s (carry slotting several under).
Running water soaks 15min or warm water (about 50 DEG C) 5min.(blotting water)
Put Yihong liquid 2min.
Conventional dehydration, transparent, mounting：95% ethanol (I) min → 95% ethanol (II) 1min → 100% ethanol (I) 1min → 100% ethanol (II) 1min → dimethylbenzene carbolic acid (3：1) 1min (omission) → dimethylbenzene (I) 1min → dimethylbenzene (II) 1min → neutral gum mounting
The results are shown in Table 5.
Table 5
Group n Pathological lesion is integrated
Blank group 20 0
Model group 20 2.11±0.47*
Growth factor control group 20 2.56±0.61*
Experimental group 1 20 1.29±0.34*△
Experimental group 2 20 1.62±0.77*△
Experimental group 3 20 1.56±0.48*△
▲ P ＜ 0.05vs blank groups；△ P ＜ 0.05vs model groups；★ P ＜ 0.05vs growth factor groups；
As shown in table 5, intraperitoneal injection CCl4After week for 7 weeks, model group and saline rats hepatic tissue pathology are damaged Integration highest, after being treated through stem cell composition of the present invention (obtained in embodiment 3~5), pathological lesion integration is remarkably decreased, with Model group is compared, and difference is statistically significant.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims (10)

1. a kind of composition, it is characterised in that including growth factor, albumin and mesenchymal stem cells MSCs.
2. composition according to claim 1, it is characterised in that the growth factor is selected from HGF, preceding Row parathyrine, insulin or hyperglycemic factor.
3. composition according to claim 2, it is characterised in that the content that the HGF accounts for composition is 30 μ g/ml~60 μ g/ml, the prostaglandin account for the content of composition for 4 μ g/ml~8 μ g/ml, the insulin account for combination The content of thing is that the content that 20U/ml~40U/ml, the hyperglycemic factor account for composition is 3mg/ml~6mg/ml.
4. the composition according to any one of claims 1 to 3, it is characterised in that the albumin accounts for the quality of composition Percentage composition is 15~30%.
5. the composition according to any one of Claims 1-4, it is characterised in that the albumin accounts for the quality of composition Percentage composition is 20%.
6. the composition according to any one of claim 1 to 5, it is characterised in that the mesenchymal stem cells MSCs it is dense Spend is 1.0~10.0 × 107/ml。
7. the composition according to any one of claim 1 to 6, it is characterised in that the mesenchymal stem cells MSCs it is dense Spend is 1.5 × 107/ml。
8. the composition according to any one of claim 1 to 7, it is characterised in that the dosage of the composition is with bone Bone marrow-drived mesenchymal stem is calculated as 1.0~10.0 × 107/ kg the weight of animals.
9. the composition according to any one of claim 1 to 8 prepare prevention and/or treatment liver diseases medicine and/ Or health products.
10. it is a kind of prevent and/or treatment liver diseases medicine and/or health products, it is characterised in that including such as claim 1 To the composition described in 8 any one.
CN201611220196.0A 2016-12-26 2016-12-26 Composition and application thereof Pending CN106692951A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104958320A (en) * 2015-07-29 2015-10-07 西安芙金细胞科技有限公司 Cell preparation for treating osteoarthritis and preparation method thereof
CN105056303A (en) * 2015-09-21 2015-11-18 广州赛莱拉干细胞科技股份有限公司 Composition, preparation and application thereof
CN105534848A (en) * 2015-12-29 2016-05-04 四川新生命干细胞科技股份有限公司 Cosmetic or pharmaceutical composition and application thereof
CN106729641A (en) * 2016-12-26 2017-05-31 广州赛莱拉干细胞科技股份有限公司 Composition and its application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104958320A (en) * 2015-07-29 2015-10-07 西安芙金细胞科技有限公司 Cell preparation for treating osteoarthritis and preparation method thereof
CN105056303A (en) * 2015-09-21 2015-11-18 广州赛莱拉干细胞科技股份有限公司 Composition, preparation and application thereof
CN105534848A (en) * 2015-12-29 2016-05-04 四川新生命干细胞科技股份有限公司 Cosmetic or pharmaceutical composition and application thereof
CN106729641A (en) * 2016-12-26 2017-05-31 广州赛莱拉干细胞科技股份有限公司 Composition and its application

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Application publication date: 20170524