CN104845932A

CN104845932A - Novel application of icariin

Info

Publication number: CN104845932A
Application number: CN201510212664.9A
Authority: CN
Inventors: 边育红; 禇晓倩; 王丽; 齐新; 李文; 崔换天; 周会芳; 赵庆云; 武玉琳
Original assignee: Tianjin Second People's Hospital; Tianjin University of Traditional Chinese Medicine; Tianjin People Hospital
Current assignee: Tianjin Second People's Hospital; Tianjin University of Traditional Chinese Medicine; Tianjin People Hospital
Priority date: 2015-04-29
Filing date: 2015-04-29
Publication date: 2015-08-19

Abstract

The invention provides a novel application of icariin. Expression of a pluripotent gene Oct-4mRNA can be improved after the icariin acts on an umbilical cord mesenchymal stem cell after a week, pluripotency of the umbilical cord mesenchymal stem cell is improved, the capacity of the umbilical cord mesenchymal stem cell differentiating a nerve cell, a myocardial cell and an islet cell can be improved, the methylation level of the pluripotent adjusting gene Oct-4 is reduced, and the acetylation level of histone H3K9 is promoted.

Description

The novelty teabag of icarin

Technical field

The present invention relates to the novelty teabag of icarin.

Background technology

Stem cell has the ability of multi-lineage potential and self, has a good application prospect in cell replacement therapy, gene therapy, organizational project etc.Research in recent years about stem cell Preclinic and clinic is advanced by leaps and bounds, make rapid progress, make people have more deep understanding to stem cell.From the research of Totipotent embryonic stem cells (ES cell), to the discovery of adult stem cell, arrive the generate induced pluripotent stem cells (iPS) that people are obtained by somatocyte reprogramming again, invariably embody researcher to the wisdom of stem-cell research and the painstaking effort of pouring into.People have responded with great enthusiasm and hope for extensive, the routine clinical application of stem cell, but the restriction of the factor such as immunological rejection, tumorigenicity, ethics morals problem due to embryonic stem cell, significantly limit its clinical application.

The focus that iPS is stem-cell research is in recent years obtained by somatocyte reprogramming, people adopt body-cell neucleus transplanting (SNCT), utilize retrovirus that specific gene (Oct-4, Nanog etc.), small molecules non-virus carrier are imported the methods such as somatocyte and all have successfully been obtained iPS.Oct-4 and Nanog is two kinds of of paramount importance transcription factors maintaining stem cell versatility and self.They only express usually in multipotential stem cell, do not express in noble cells.Adopt at present inoblast more, epidermic cell carry out inverse differentiation as donorcells and obtain multipotential cell, these research work demonstrate cell against point voltinism.But inoblast, epidermic cell are terminally differentiated cells, it is poor against differentiation capability, the time of reprogrammed required for multipotential cell is long, efficiency is low.Current research shows, what application had that the adult stem cell of certain stem cell properties can be more prone to obtains iPS, as reports such as Kim: neural stem cell can simplify reprogrammed step and obtain iPS; Research simultaneously shows, external application micromolecular compound can increase somatocyte against differentiation ratio, improves inverse differentiation efficiency.

But, present stage some disease of adult cell transplantation treatment used, but adult cell versatility is poor, affects result for the treatment of, and the scheme of present traditional inducing cell versatility is virus or genetic modification, and the cell of process is like this unfavorable for that transplanting is to people or animal.

Human umbilical cord mesenchymal stem cells (hUMSCs) is that people are separated from fetal cord Wal Tong Shi glue (Whartonps Jelly) position the stroma cell obtained in recent years.HUCMSCs has certain stem cell properties, and its multi-lineage potential and plasticity-are confirmed.Under specified conditions, hUCMSCs can be divided into scleroblast, chondrocyte, adipocyte, islet cells, neurocyte, myocardial cell and sexual cell etc.In view of umbilical cord mesenchyma there is certain differentiation versatility, plasticity-is strong, rejection is low, without advantages such as ethics disputes, hUMSCs will be expected to become autologous with allotransplantation and the desirable cell derived of cell replacement therapy.Although hUCMSCs has certain stem cell properties, its differentiation capability still has certain limitation.If drug intervention can be utilized to induce hUCMSCs, make it obtain versatility as ES, then can avoid applying immunological rejection, ethics problem and the shortcoming such as Operating Complexity, tumorigenicity of application retrovirus induction caused by iPS that embryonic stem cell produces.

Traditional Chinese medicine " kidney storing essence " theory thinks that the prosperity and decline of kidney essense decides the growth of human body, growth and reproduction, the ability that its intension and function and stem cell have Multidirectional Differentiation and self duplication is closely related, and many kidney-nourishing and essence-enriching herbs have the function promoting differentiation of stem cells and propagation.Given this, the present invention for point of penetration, by observing the impact that conventional kidney-nourishing tcm drug effective constituent is expressed hUMSCs versatility regulatory gene Oct-4, filters out the kidney-nourishing tcm drug activeconstituents affecting hUMSCs versatility with Oct-4 versatility regulatory gene; By investigating, the activeconstituents filtered out is inside on hUMSCs, the impact of domestic and abroad triploblastica cytodifferentiation, the effect of confirmation kidney-nourishing tcm drug activeconstituents induction hUMSCs versatility and using value.

In many kidney-nourishing tcm drug effective constituent, icarin is the over-ground part of Berberidaceae plant Herba Epimedii (Epimediumgrandiflorum Morr.), and molecular formula is C ₃₃h ₄₀o ₁₅, molecular weight 676.65 is faint yellow needle crystal, fusing point 231-232 DEG C.Be dissolved in ethanol, ethyl acetate, be insoluble to ether, benzene, chloroform.Icarin has effects such as promoting hemopoietic function, immunologic function and bone metabolism, modern pharmacology experimental study shows: Herba Epimedii can increase cardiovascular and cerebrovascular volume of blood flow, promote hemopoietic function, immunologic function and bone metabolism, there is the effects such as anti-ageing, antitumor, be used to the application and induce in the research of hUMSCs versatility.

Summary of the invention

Technical problem to be solved by this invention is the novelty teabag providing icarin.

For solving the problems of the technologies described above, technical scheme of the present invention is:

Icarin is strengthening the purposes in stem cell versatility.

Preferably, the purposes of above-mentioned icarin, by icarin process stem cell, improves stem cell versatility, then transplants stem cell to animal.

Preferably, the purposes of above-mentioned icarin, is applied to the nutrient solution treating transplanted cells when inducing culture is used for the treatment of acute liver damage or cerebral ischemia.

Preferably, the purposes of above-mentioned icarin, the method for described inducing culture cell is: umbilical cord mesenchymal stem cells cultivates 7 days in the perfect medium containing 100 μMs of icarin, and within every 3 days, change liquid once, culture condition is 37 DEG C, 5%CO ₂obtain the stem cell that versatility strengthens, described perfect medium is DMEM-F12 substratum+20%FBS (foetal calf serum)+1%LG (glutamine)+1%PS (penicillin streptomycin)+10ng/mlEGF (Urogastron)+10ng/mlbFGF (Basic Fibroblast Growth Factor).

The invention has the beneficial effects as follows:

Icarin effect umbilical cord mesenchymal stem cells can strengthen its versatility in one week, promote its neuralward cell, myocardial cell, the differentiation of islet cells, specify that the external ability had to triploblastica cytodifferentiation of hUMSCs (human umbilical cord mesenchymal stem cells), experiment in vivo sets up the damage model representing three germinal layer Different Organs, transplant the hUMSCs after ICA intervention, by the every function criterion of evaluation model animal and transplanted cells differentiation capability situation, demonstrate kidney-nourishing tcm drug activeconstituents breaks up versatility impact on hUMSCs further.Simultaneously by studying icarin to the change of umbilical cord mesenchymal stem cells in genome integral level; show that icarin effect umbilical cord mesenchymal stem cells is after one week, versatility regulatory gene Oct-4 methylation level can be reduced and promote histone H 3 K9 Acetylation Level.

Verify by experiment, net result shows that icarin can strengthen its versatility after acting on umbilical cord mesenchymal stem cells, disclose the Molecular Biology Mechanism that icarin acts on umbilical cord mesenchymal stem cells, provide theoretical and experimental basis for umbilical cord mesenchymal stem cells is widely used in regenerative medicine.

Accompanying drawing explanation

Fig. 1 is the primary of hUCMSCs and Secondary Culture morphological change figure (inverted microscope, × 100), a: original cuiture 5 days, b: original cuiture one week, c: original cuiture two weeks, d: Secondary Culture P1;

Fig. 2 is Immunofluorescence coloration result figure,

a:CD29(×400),b:CD44(×400),c:CD90(×200)；d:C45(×200),e:CD31(×200),f:CD34(×200)；

Fig. 3 is Flow cytometry human umbilical cord mesenchymal stem cells phenotypic map;

Fig. 4 is that icarin raises human umbilical cord mesenchymal stem cells versatility gene Oct-4 expression figure,

*P<0.05.**P<0.01；

Fig. 5 is neuralward like cell inducing morphological variation diagram,

A: control group (× 100); B: induction 4h (× 100); C: induction 6h (× 200);

Fig. 6 is identified by immunofluorescence result figure,

A: control group, b:nestin, c:GFAP, d:Map-2 (× 200);

Fig. 7 is cells into cardiomyocytes inducing morphological variation diagram,

A: control group (× 200); B: induce 4 weeks groups (× 200);

Fig. 8 is that RT-PCR detects cTNI mrna expression result figure,

A: reference gene GAPDH; B: goal gene cTNI; 1: do not induce group; 2: induction group;

Fig. 9 is to islet cells inducing morphological variation diagram,

A does not induce group (× 100), and b induces 12 days groups (× 100);

Figure 10 is dithizone coloration result figure,

A: do not induce group (× 200), b: induce 12 days groups (× 200);

Figure 11 is Transplanted cells 3 days to the effect diagram of Mouse Blood creatinine and blood urea nitrogen;

Figure 12 is Transplanted cells 7 days to the effect diagram of Mouse Blood creatinine and blood urea nitrogen;

Figure 13 be living imaging system to the external result figure that takes pictures of nephridial tissue---Transplanted cells 3d and 7d nephridial tissue fluorescence signal intensity, remarks: nephridial tissue order is successively control group, model group, MSC group, MSC+I group;

Figure 14 is Transplanted cells 3 days and 7 days nephridial tissue fluorescence signal intensity figure;

Figure 15 is impact (× 100) figure of Transplanted cells 3d on Renal Morphology;

Figure 16 is impact (× 100) figure of Transplanted cells 7d on Renal Morphology;

Figure 17 is that Transplanted cells 3d hUCMSCs breaks up situation (× 200) figure to renal cells, and wherein, red fluorescence is that DIR marks hUCMSCs, green fluorescence is renal cells marker Cadherin, blue-fluorescence is DAPI;

Figure 18 is that Transplanted cells 7d hUCMSCs breaks up situation (× 200) figure to renal cells, and wherein, red fluorescence is that DIR marks hUCMSCs, green fluorescence is renal cells marker Cadherin, blue-fluorescence is DAPI;

Figure 19 is tetracol phenixin modeling 24 hours effect diagram to mouse ALT and AST;

Figure 20 is Transplanted cells 3 days to the effect diagram of mouse gpt and glutamic-oxal(o)acetic transaminase;

Figure 21 is Transplanted cells 7 days to the effect diagram of mouse gpt and glutamic-oxal(o)acetic transaminase;

Figure 22 is transplanted cells 3 days and 7 days effect diagram to liver coefficient;

Figure 23 is Transplanted cells 3d and 7d hepatic tissue fluorescence signal intensity figure, remarks: hepatic tissue order is successively control group, model group, MSC group, MSC+I group;

Figure 24 is Transplanted cells 3 days and 7 days hepatic tissue fluorescence signal intensity figure;

Figure 25 is impact (× 100) figure of Transplanted cells 3d on liver morphology;

Figure 26 is impact (× 100) figure of Transplanted cells 7d on liver morphology;

Figure 27 be Transplanted cells 3d hUCMSCs to hepatocyte differentiation situation (× 200) figure, remarks: red fluorescence is that DIR marks that hUCMSCs, green fluorescence are liver cell marker AFP, blue-fluorescence is DAPI;

Figure 28 be Transplanted cells 7d hUCMSCs to hepatocyte differentiation situation (× 200) figure, remarks: red fluorescence is that DIR marks that hUCMSCs, green fluorescence are liver cell marker AFP, blue-fluorescence is DAPI;

Figure 29 is the effect diagram that transplanted cells is expressed 2-VO rat model hippocampus Nestin;

Figure 30 is the effect diagram that transplanted cells is expressed 2-VO rat model hippocampus Map-2;

Figure 31 be after ICA intervenes Nanog the variation diagram of Oct-4 gene promoter region DNA methylation;

Figure 32 is the change scattergram of Nanog, Oct-4 gene promoter region DNA methylation, and in figure, stain shows methylated CpG dinucleotides, and white point is the CpG dinucleotides of demethylation;

Figure 33 is Oct-4 gene promoter region acetylation of histone data.

Embodiment

In order to make those skilled in the art better understand technical scheme of the present invention, below in conjunction with embodiment, technical scheme of the present invention is described in further detail.Used by wherein, in embodiment:

Icarin is purchased from institute for drug control, Tianjin.

Perfect medium: DMEM-F12 substratum+20%FBS (foetal calf serum)+1%LG (glutamine)+1%PS (penicillin streptomycin)+10ng/mlEGF (Urogastron)+10ng/mlbFGF (Basic Fibroblast Growth Factor)

Embodiment 1

1, the Isolation and identification of hUMSCs

Separating funicle mesenchyme stem cell: aseptically umbilical cord 2% dual anti-D-hank ' s is rinsed 2-3 time repeatedly, remove remained blood as far as possible, and reject artery and vein vascular.Umbilical cord after cleaning is placed in glass dish, is cut into 1mm ³size, adds Collagenase I and collagenase II (final concentration is 0.05%), is placed in 37 DEG C of constant-temperature tables, digestion 30min.Scalping is filtered, and filtrate is A, and residue tissue adds 0.25% pancreatin and continues digestion 30min; Add FBS after digestion bundle and stop digestion, digestion product filters through wide-meshed screen, liquor B.Mixed by A, B liquid, then filter through 200 eye mesh screens, collect filtrate, 1000rpm, 15min, add full training (DF12+20%FBS+1%L-G+1%PS+10ng/mlEGF+10ng/mlbFGF), is placed in 37 DEG C of saturated humidities, 5%CO ₂incubator is cultivated.Within one week, change liquid twice, when cytogamy is to about 80-90%, go down to posterity.The forth generation hUCMSCs of experiment stable growth.

Qualification umbilical cord mesenchymal stem cells: the 4th generation hUCMSCs is got in experiment, utilizes 0.25% trypsinase-0.02%EDTA to digest, by 2 × 10 ⁴the density in/hole is inoculated in 12 orifice plates wrapped in advance by cover glass, 5%CO ₂, cultivate in 37 DEG C of incubators, after adherent 24h, fix 10min with 4% paraformaldehyde, PBS washes 3 times, normal sheep serum closes 30min, adds primary antibodie (CD29, CD44, CD90, CD31, CD34, CD45) ratio 1:50 (antibody diluent is joined), and not adding primary antibodie does negative control simultaneously, 4 DEG C are spent the night, after washing 3 times with PBS every other day, add two and anti-hatch 1h, discard after PBS washes 3 times, add DAPI and contaminate core, fluorescence microscopy Microscopic observation is also taken pictures.

The 4th generation hUCMSCs is got in experiment, selects anti-human CD29, CD44, CD90, CD31, CD34, CD45 antibody respectively, and application Flow Cytometry is identified isolated cell.

2, experimental result

Separation and Culture hUCMSCs: original cuiture five days rear visible several spindle shapes become fibrous cell attachment to grow, after one week, visible cell increases gradually, after two weeks, cytogamy is to more than 90%, typical spindle shape, and become swirling to grow, namely can go down to posterity, after going down to posterity, the even adherent growth of cell, is shown in Fig. 1.

Primary qualification hUCMSCs: Immunofluorescence dyeing and flow cytometry results display, hUCMSCs expresses mesenchyme dry surface antigen CD29, CD44, CD90 is positive, sees Fig. 2 .a, b, c), and does not express CD31, CD34, CD45, be shown in Fig. 2 .d, e, f.Flow cytometry is surveyed and be the results are shown in Figure 3.

Experimental result shows that this is tested separation method used and can obtain hUCMSCs, and cell purity is higher, and cultural method is reliable, and upgrowth situation is good, can carry out stable Secondary Culture, can be used for subsequent experimental.

Embodiment 2

Kidney-nourishing tcm drug activeconstituents is on the impact of hUCMSCs versatility

1, different kidney-nourishing tcm drug activeconstituents impact that hUCMSCs versatility gene Oct-4 is expressed

Select conventional kidney-nourishing tcm drug activeconstituents: icarin, Herba Epimedii total flavones, semen cuscutae flavonoids, morindea officinalis polysaccharide, naringin, loganin and morroniside, wherein, morindea officinalis polysaccharide, Herba Epimedii total flavones and semen cuscutae flavonoids are for certainly proposing acquisition, and all the other are all buy gained mark product.Wherein,

Morindea officinalis polysaccharide extracting method is: precision takes Root of Medicinal Indian mulberry medicinal material, pulverizes, adds 10 times of volume water supersound extraction, recycle-water extract evaporate to dryness, adds 75% EtOH Sonicate mixing, hold over night, remove ethanol, reclaim precipitation, extraction into ethyl acetate removing oil-soluble impurities, reclaim water layer, Sevage method removing protein, is recycled to dry, activated carbon decolorizing, filtrate is recycled to 10-20ml, leaves standstill after 80% EtOH Sonicate, removing ethanol, obtains morindea officinalis polysaccharide;

Herba Epimedii total flavones extracting method is: precision takes epimedium herb, pulverizes, 95% EtOH Sonicate 40min, the outstanding dry recovery of extracting liquid filtering, products in water disperses, the petroleum ether degreasing of 3 times of volumes, obtains water layer propyl carbinol (1:1) extraction, obtain crude extract medicinal extract, get macroporous resin column, wet method loading, after washing 5 volumes, use 20% and 60% ethanol elution respectively, reclaim and obtain Herba Epimedii total flavones;

Semen cuscutae flavonoids extracting method is: precision takes Semen Cuscutae, pulverizes, 95% EtOH Sonicate 40min, the outstanding dry recovery of extracting liquid filtering, products in water disperses, the petroleum ether degreasing of 3 times of volumes, obtains water layer propyl carbinol (1:1) extraction, obtain crude extract medicinal extract, get macroporous resin column, wet method loading, after washing 5 volumes, use 10% and 30% ethanol elution respectively, reclaim and obtain excessive semen cuscutae flavonoids.

Above-mentioned activeconstituents is divided into high, medium and low dosage group, concrete dosage is as follows: the high, medium and low dosage of icarin, naringin, loganin, morroniside is respectively 100uM, 10uM and 1uM, the high, medium and low dosage of Herba Epimedii total flavones, semen cuscutae flavonoids, morindea officinalis polysaccharide is respectively 50ug/ml, 5ug/ml and 0.5ug/ml, arranges blank group simultaneously.The well-grown hUCMSCs of forth generation is got in experiment, and the trypsinase with 0.25% and the digestion of 0.02%EDTA mixed solution, be dispersed into unicellular by hUCMSCs digestion, and adjustment cell density is 5 × 10 ⁴mL ^-1be inoculated in six orifice plates, every hole 2mL, after cell attachment, add after said medicine acts on 72h respectively, collecting cell, extracts total serum IgE, and adopt RT-PCR method, detection of drugs is on the impact of versatility regulatory gene Oct-4 mrna expression level.Filter out effective dose and the time of the activeconstituents affecting hUCMSCs versatility.

2, icarin impact that hUCMSCs versatility gene Oct-4 is expressed

The well-grown hUCMSCs of forth generation is got in experiment, and the trypsinase with 0.25% and the digestion of 0.02%EDTA mixed solution, be dispersed into unicellular by hUCMSCs digestion, and adjustment cell density is 5 × 10 ⁴mL ^-1be inoculated in six orifice plates, every hole 2mL, after cell attachment, add after icarin 100uM acts on 72h and 1w respectively, collecting cell, extracts total serum IgE, adopt RT-PCR method, detect icarin 100uM to the impact of versatility regulatory gene Oct-4 mrna expression level.

3, Q-PCR verifies the impact that icarin is expressed hUCMSCs versatility gene Oct-4

The well-grown hUCMSCs of forth generation is got in experiment, and the trypsinase with 0.25% and the digestion of 0.02%EDTA mixed solution, be dispersed into unicellular by hUCMSCs digestion, and adjustment cell density is 5 × 10 ⁴mL ^-1be inoculated in six orifice plates, every hole 2mL, after cell attachment, add after icarin 100uM10uM and 1uM act on 72h and 1w respectively, collecting cell, extracts total serum IgE, adopt Q-PCR method, detect icarin 100uM to the impact of versatility regulatory gene Oct-4mRNA expression level.

4, experimental result

First the impact adopting the method for RT-PCR to investigate 7 taste kidney-nourishing tcm drug effective constituents to express versatility gene oct-4.After having acted on 72 hours, the compositions such as icarin, Herba Epimedii total flavones, morindea officinalis polysaccharide all can improve Oct-4 genetic expression, and wherein icarin expression level is higher than other components, sees Fig. 4-A.Drug treating time is brought up to 1 week, adopt the impact that RT-PCR method investigation icarin is expressed versatility gene oct4, experimental result draws, after icarin 100 μMs acts on 72h and 1W, versatility gene oct4 is expressed and all there is significant difference (p<0.05), see Fig. 4-B.For icarin group, further employing Q-PCR method is verified, result show that icarin 100 μMs all can significantly improve versatility gene oct4 at 72 and 1 weeks two time points and express, and 1 week time, there is pole significant difference, see Fig. 4-C, therefore follow-up test adopts icarin 100 μMs as administration condition.

Embodiment 3

Kidney-nourishing tcm drug activeconstituents is on the impact of umbilical cord mesenchymal stem cells to triploblastica histocyte differentiation capability

I in vitro study---hUMSCs is to the impact of triploblastica histocyte differentiation capability

1, external evoked hUCMSCs differentiating into nerve cells: the 4th generation hUCMSCs is got in experiment, 0.25% trypsinase-0.02%EDTA is utilized to digest, cell climbing sheet, when Growth of Cells converges to 50%-60%, after first using low sugar DMEM (10%FBS) pre-induced 24 h containing 1 mmol/L beta-mercaptoethanol, suck pre-induced liquid, PBS washs 3 times, the serum-free low sugar DMEM/F12 being 200 μm of ol/L BHA BHA with 2% dimethyl sulfoxide (DMSO) and final concentration again carries out induction and hatches, observation of cell metamorphosis under inverted microscope after 4-6h.

2, Immunofluorescence detects neural-like cells surface marker: get the cell after induction, 4% paraformaldehyde fixes 10min, PBS washes 3 times, normal sheep serum closes 30min, add primary antibodie GFAP (1:100), MAP-2 (1:100), Nestin (1:50), not adding primary antibodie does negative control simultaneously, 4 DEG C are spent the night, after washing 3 times with PBS every other day, add two and anti-hatch 1h, discard after PBS washes 3 times, add DAPI and contaminate core, fluorescence microscopy Microscopic observation is also taken pictures.

3, external evoked hUCMSCs myocardiac differentiation: the 4th generation hUCMSCs is got in experiment, utilizes 0.25% trypsinase-0.02%EDTA to digest, according to 5 × 10 ⁴the density in/hole is inoculated in 6 orifice plates, 5%CO ₂, cultivate in 37 DEG C of incubators, until thin adherent culture after 2 days, after induction group adds 10 μMs of U-18496+DMEM/F12 culture medium culturing 24h, be changed to normal perfect medium and cultivate 4 weeks, control group is that perfect medium is cultivated all the time.

4, RT-PCR detects cTnC TNI mrna expression: cell induction is after 4 weeks, PBS washes three times, add RTIZol lysing cell, extract total serum IgE according to RNA simple Total RNA kit specification sheets, RT-PCR step is carried out according to Quantscript RT Kit.

5, external evoked hUCMSCs is to islet cell differentiation: the 4th generation hUCMSCs is got in experiment, utilizes 0.25% trypsinase-0.02%EDTA to digest, is inoculated in 12 orifice plates, 5%CO according to the density in 2 × 104/ holes ₂cultivate in 37 DEG C of incubators, after cytogamy is to about 80%, induction group adds first stage inductor: containing 0.55 μm of ol/L beta-mercaptoethanol+DMEM in high glucose culture medium culturing after 2 days, be changed to subordinate phase inductor: 100ng/mlEGF+10ng/mlbFGF+2%B27+ DMEM in high glucose culture medium culturing is after 6 days, be changed to phase III inductor: 20mmol/L nicotinamide+DMEM in high glucose culture medium culturing 4 days, control group is that perfect medium is cultivated all the time.

6, dithizone dyeing: after the induction cell of 12 days and the cell PBS that do not induce are washed 3 times, every hole adds 1 mL1% dithizone (PBS joins), puts in 37 DEG C of incubators and hatches 20 min, with inverted microscope observation of cell coloring case.

MAIN OUTCOME MEASURES: the 1. form of the primary separation and Culture of umbilical cord mesenchymal stem cells.2. the cell fluorescence chemical coloration result of umbilical cord mesenchymal stem cells surface markers.3. the metamorphosis and the RT-PCR that are induced to differentiate into myocardial cell detect cTNI mrna expression situation.4. metamorphosis and the dithizone coloration result of islet cells is induced to differentiate into.

7, experimental result:

7.1 neuralward like cell inducing morphological changes: after cell adds pre-induced liquid 24 h, part cell starts the death that suspends, remaining cell form retraction becomes irregular, formal induction 4h rear section cell cytoplasm bounces back to core, it is strong that cell space becomes periphery refractivity, sees Fig. 5 b (Fig. 5 a is control group).After inducing 6 h, tenuigenin shrinks centered by nucleus, and cell shortens, smaller volume, and cell forms Cellbody dendrite that is bipolar or multipole, occurs similar neurocyte form, sees Fig. 5 c.

7.2 Immunofluorescences detect neural-like cells surface marker: result shows cell expressing GFAP and MAP-2 after inducing 6h, but does not express Nestin, and control group is not expressed, as shown in Figure 6.

7.3 cells into cardiomyocytes inducing morphological changes: after cell induction 24h, small portion necrocytosis, most cells continues adherent growth, and after induction 7d, Growth of Cells is slow, and form expands, and occurs flesh filamentary texture, as shown in Figure 7 after inducing 4 weeks.

7.4RT-PCR detects cTNI mrna expression result: the cell of not inducing low expression cTNI gene, induction group is strongly expressed cTNI gene after 12 days, as shown in Figure 8.

7.5 change to islet cells inducing morphological: after first stage induction, part necrocytosis, remaining cell adherent growth, after subordinate phase induction, cellular form becomes three-dimensional slender type, after phase III induction, cell bounces back, and graduates into spherical and assembles agglomerating, as shown in Figure 9.

7.6 dithizone coloration results: observing nearly all cell under induction group inverted microscope is red-brown, and the cell of not inducing is not painted, as shown in Figure 10.

Visible, human umbilical cord mesenchymal stem cells neuralward cell direction, before induction, human umbilical cord mesenchymal stem cells is typical spindle shape, and becomes swirling to grow; After the combined inductions such as BHA, cellular form changes trend neuron, its expression of Immunofluorescence test microtubule-associated protein MAP2 and glial fibrillary acidic protein GFAP; Cardiocytes direction, after U-18496 induction after one or two weeks, cell volume becomes large, poor growth, induce RT-PCR after 4 weeks to detect its myocardial cell surface mark troponin cTnI expression, induction group cTnI mrna expression level does not induce group obviously to increase; To insulin-like cell direction, through combined inductions such as beta-mercaptoethanols after 12 days, induction group cell becomes round gradually, and assembles agglomerating, and dithizone coloration result display induction group cell more than 90% is induced successfully.In sum, human umbilical cord mesenchymal stem cells has multi-lineage potential, is externally induced to differentiate into neural-like cells, cardiac-like muscle cell and insulin-like cell.

II In vivo study---investigate kidney-nourishing tcm drug activeconstituents to the impact of hUMSCs to triploblastica histocyte differentiation capability

1. kidney-nourishing tcm drug activeconstituents intervenes the impact of umbilical cord mesenchymal stem cells transplanting on acute injury of kidney

1, cell dosing and mark

HUCMSCs after going down to posterity is inoculated in the culturing bottle of T75, density is 4 × 10 ⁴/ ml, 15ml, adds the icarin perfect medium containing 100 μMs next day, cultivates 7 days altogether, and liquid is changed once in 3 days in centre, when cell grows to 80% ~ 90% fusion, 0.25% trypsinase and 0.02%EDTA peptic cell.Umbilical cord mesenchymal stem cells after the induction of DIR mark.

2, acute injury of kidney (AKI) model

Cisplatin injections normal saline dilution becomes 1mg/ml, with 11.5mg/kg dosage abdominal injection, sets up AKI model.

3, rear Transplanted cells set up by mouse cis-platinum acute injury of kidney model

After utilizing cis-platinum modeling 24h, control group and model group are through tail venoclysis physiological saline 0.4ml; MSC group: the normal MSC cell suspension 4 × 10 marked through tail venoclysis DIR ⁶/ 0.4ml; MSC+I group: through tail venoclysis DIR mark and through icarin intervene MSC cell suspension 4 × 10 ⁶/ 0.4ml.

Testing index:

Respectively at after transplanting MSC the 3rd and 7 days random each group mouse 5, the eye corner of the eyes gets blood, is placed in water bath, 37 DEG C of water-bath half an hour, separation of serum after the centrifugal 15min of 3000rpm ,-20 DEG C of cryopreservation, for subsequent use, after sample set is neat, rewarming semi-automatic biochemical analyzer measures serum creatinine and blood urea nitrogen.

Cut bilateral renal, after small animal living body imaging system is taken pictures rapidly, cut its tunicle, stringer is cut open, and left side nephridial tissue is fixed with the formalin of 10% and numbers, paraffin bag quilt, 6 μm of thick serial section, and routine does HE dyeing, light Microscopic observation.Observe in 10 visuals field that light Microscopic observation is non-overlapped often opening section, injury of renal tubular is defined as: renal tubular necrosis or brush border come off, cast is formed, lumen distention.

The permanent frozen sheet cutter section of right side nephridial tissue, thick about 10 μm of Fluirescence observation sheet, carries out immunohistochemistry's dyeing observation and detects hUCMSCs to renal cells differentiation situation etc.

Cell is transplanted to after in Mice Body, respectively at time point be 3 days and 7 days time, the often random taking-up of group three mouse kidneys, by the distribution situation of small animal living body imager vitro detection transplanted cells at kidney.

4, experimental result

4.1 Transplanted cellss are on the impact of AKI Mouse Blood creatinine and blood urea nitrogen

From table 1 and Figure 11, Transplanted cells, after 3 days, is compared with Normal group, and model group experiment mice serum creatinine and urea nitrogen levels (BUN and CRE) all significantly raise (P < 0.01), modeling success;

Compare with model group, after vein transplantation MSC and icarin intervene MSC, BUN level decreases, the downtrending of MSC+I group is obvious, but unknown significance difference, after Transplanted cells, CRE level obviously reduces, and MSC+I group is more remarkable and have notable difference (P < 0.05).

From table 2 and Figure 12, Transplanted cells, after 7 days, is compared with Normal group, and model group experiment mice BUN and CRE level all significantly raise (P < 0.01), and MSC group and MSC+I group BUN and CRE have no notable difference;

Compare with model group, MSC group and MSC+I group all have the trend that can reduce BUN and CRE level, and MSC group and MSC+I group can reduce CRE level (P < 0.05), compared with MSC group, MSC+I group BUN and CRE level decrease, but have no notable difference.

Table 1 Transplanted cells 3 days on the impact of Mouse Blood creatinine and blood urea nitrogen (n=5, )

* P < 0.05 is compared with normal group; * compares P < 0.01 with normal group;

# compares P < 0.05 with model group; ## compares P < 0.01 with model group;

△ and MSC group compares P < 0.05; △ △ compares P < 0.01 with MSC group;

Table 2 Transplanted cells 7 days on the impact of Mouse Blood creatinine and blood urea nitrogen (n=3, )

# compares P < 0.05 with model group; ## compares P < 0.01 with model group;

△ and MSC group compares P < 0.05; △ △ compares P < 0.01 with MSC group;

The impact that 4.2 icarin interventions are moved hUMSCs vein transplantation

Small animal living body imaging system takes pictures result as shown in Figure 13,14 and table 3, Transplanted cells 3 and after 7 days, and put to death animal and take out kidney rapidly, MSC group and MSC+I group all have red fluorescent, and MSC+I group has the trend of enhancing than MSC group fluorescence intensity.

Table 3 Transplanted cells 3 days and 7 days nephridial tissue fluorescence signal intensities (n=3, )

* P < 0.05 is compared with MSC group; * * and MSC group compares P < 0.01;

4.3 Transplanted cellss are on the impact of kidney of mouse form

The change of the 3rd day MSC group and MSC+I group renal histology after Microscopic observation model group and Transplanted cells.The visible tubular ectasia of AKI model group, contour structure is unintelligible, cell arrangement is mixed and disorderly, part Tubular epithelial cell vacuolar degeneration and swelling, the formation of cast, and interstitial inflammatory cells infiltrates and congested.Transplanted cells group epithelial cell marshalling also has a small amount of cavity to be out of shape, but tubular formation is less, with MSC+I better effects if, sees Figure 15;

Transplant latter 7th day, mouse AKI model group uriniferous tubules is still expanded, epithelial cell shedding, inflammatory cell infiltration.MSC group tubular ectasia alleviates, and is tending towards normal, with MSC+I better effects if, sees Figure 16.

4.4 immunofluorescence methods detect hUCMSCs and break up situation to renal cells

Immunofluorescence method result is as shown in Figure 17,18: the hUCMSCs that MSC group and MSC+I group all have a small amount of DIR red fluorescence to mark expresses renal cells surface marker Cadherin (green fluorescent label) simultaneously, mostly be the region that several cell aggregation is closing on, illustrate that the hUCMSCs transplanted can break up to renal cells, but have no notable difference between MSC group and MSC+I group.

2. kidney-nourishing tcm drug activeconstituents intervenes the impact of umbilical cord mesenchymal stem cells transplanting on acute liver damage

1, cell dosing and mark:

2, acute liver damage (ALI) model is made:

0.2%CCl ₄, with 10 ml/kg dosage abdominal injections, set up ALI model, Normal group is to the sweet oil of equivalent.

3, mouse carbon tetrachloride acute hepatic injury model sets up the transplanting of rear MSC:

After utilizing tetracol phenixin modeling 24h, control group and model group are through tail venoclysis physiological saline 0.4ml; Umbilical cord mesenchymal stem cells (MSC group): the normal MSC cell suspension 4 × 10 marked through tail venoclysis DIR ⁶/ 0.4ml; Icarin intervenes umbilical cord mesenchymal stem cells (MSC+I) group: the dosing MSC cell suspension 4 × 10 marked through tail venoclysis PKH26 ⁶/ 0.4 ml.

4, Testing index:

Respectively at the 3rd day and 7 days random each group mouse 5 after transplanting MSC, the eye corner of the eyes gets blood, is placed in water bath, 37 DEG C of water-bath half an hour, sucking-off serum after the centrifugal 15min of 3000rpm ,-20 DEG C of cryopreservation, for subsequent use, after sample set is neat, rewarming semi-automatic biochemical analyzer measures Serum ALT and AST.

After extracting blood, cut liver rapidly, weigh, with normal saline flushing, filter paper blots, and weighs, and calculates liver coefficient.Organ coefficient (%)=organ weights (g)/body weight (g) × 100%;

Left lobe of liver tissue is fixed with the formalin of 10% and numbers, section, thick about 6 μm, and HE dyes, and observes, pathology of hepar inspection and evaluation hepar damnification situation examination histological change to often opening section in 10 visuals field that light Microscopic observation is non-overlapped.

Right lobe of liver organizes permanent frozen sheet cutter to cut into slices, thick about 10 μm of Fluirescence observation sheet, carries out immunohistochemistry's dyeing and fluorescence microscope and detects hUCMSCs to hepatocyte differentiation situation etc.

Cell is transplanted to after in Mice Body, respectively at time point be 3 days and 7 days time, the often random taking-up of group three mouse livers, by the distribution situation of small animal living body imager vitro detection transplanted cells at liver.

5, experimental result

5.1 model elaborates

After utilizing tetracol phenixin modeling 24h, get 3 normal mouse model mices respectively at random, the eye corner of the eyes gets blood, separation of serum, semi-automatic biochemical analyzer detects ALT and AST, as shown in Table 7, compared with normal group mouse, model group ALT and AST level significantly raise (P < 0.01), prove that model is successfully established, and the results are shown in Table 4 and Figure 19.

The table 4 tetracol phenixin modeling impact on mouse ALT and AST in 24 hours (n=3, )

5.2 Transplanted cellss are on the impact of mouse gpt and glutamic-oxal(o)acetic transaminase

From table 5-6 and Figure 20-21, the level of ALI mouse gpt and glutamic-oxal(o)acetic transaminase was not affected in after transplanted cells 3 days and 7 days.

Table 5 Transplanted cells 3 days on the impact of mouse gpt and glutamic-oxal(o)acetic transaminase (n=5, )

# compares P < 0.05 with model group; ## compares P < 0.01 with model group;

△ and MSC group compares P < 0.05; △ △ compares P < 0.01 with MSC group;

Table 6 Transplanted cells 7 days on the impact of mouse gpt and glutamic-oxal(o)acetic transaminase (n=5, )

# compares P < 0.05 with model group; ## compares P < 0.01 with model group;

△ and MSC group compares P < 0.05; △ △ compares P < 0.01 with MSC group

5.3 Transplanted cellss are on the impact of mouse liver coefficient

As shown in Table 7: compared with control group, Transplanted cells 3 days, model group and MSC group liver coefficient have significance to raise (P < 0.01), and MSC+I group also has rising (P < 0.05); Compared with model group, MSC group and MSC+I group have the trend reducing liver coefficient; Transplanted cells is after 7 days, MSC group and MSC+I group, liver coefficient and model group and normal group there was no significant difference.

Table 7 transplanted cells 3 days and the impact on liver coefficient in 7 days (n=5, )

# compares P < 0.05 with model group; ## compares P < 0.01 with model group;

△ and MSC group compares P < 0.05; △ △ compares P < 0.01 with MSC group

The impact that 5.4 icarin interventions are moved hUMSCs vein transplantation

After Transplanted cells 3 and 7 days, put to death animal and take out liver rapidly, application small animal living body imaging system detects transplanted cells and arrives lesions position situation.Small animal living body imaging system to the external result of taking pictures of hepatic tissue as shown in table 8 and Figure 23-24: after transplanting, MSC group and MSC+I group all detect red fluorescent, compared with MSC group, Transplanted cells 3 days, the fluorescent signal stronger (P < 0.05) of MSC+I group; Transplanted cells 7 days, the fluorescent signal of MSC+I group is obviously better than MSC group (P < 0.01)

Table 8 Transplanted cells 3 days and 7 days hepatic tissue fluorescence signal intensities (n=4, )

* P < 0.05 is compared with MSC group; * * and MSC group compares P < 0.01;

5.5 Transplanted cellss are on the impact of ALI mouse liver tissue morphology

As shown in figure 25, Normal group mouse liver color is sorrel, and matter is soft, smooth surface, glossy; Conventional H E dyes visible liver lobule structural integrity, radially arranges around central vein.Transplanted cells 3d, the HE of model group mouse liver dye around central vein and occur ballooning degeneration of liver cells or steatosis, visible inflammatory cell infiltration, and MSC group and the above-mentioned change of MSC+I group are also not obvious;

As shown in figure 26, extending to 7d along with the time, still there is above tissue change in model group, but all has and alleviate, and MSC group and MSC+I group liver structure recover normal substantially completely.

5.6 immunofluorescence methods detect hUCMSCs to hepatocyte differentiation situation

Immunofluorescence method detect transplant hUCMSCs to hepatocyte differentiation situation result as shown in Figure 27-28: the hUCMSCs that MSC group and MSC+I group all have a small amount of DIR red fluorescence to mark expresses surface of hepatocytes marker AFP (green fluorescent label) simultaneously, mostly be the region that several cell aggregation is closing on, illustrate that the hUCMSCs transplanted to hepatocyte differentiation, but can have no notable difference between MSC group and MSC+I group.

3. kidney-nourishing tcm drug activeconstituents intervenes the impact of umbilical cord mesenchymal stem cells transplanting on cerebral ischemia

1, cell dosing and mark:

HUCMSCs after going down to posterity is inoculated in the culturing bottle of T75, density is 4 × 10 ⁴/ ml, 15ml, adds the icarin perfect medium containing 100 μMs next day, cultivates 7 days altogether, and liquid is changed once in 3 days in centre, when cell grows to 80%-90% fusion, 0.25% trypsinase and 0.02%EDTA peptic cell.Umbilical cord mesenchymal stem cells after the induction of DIR mark.

2, the preparation of rat brain 2-VO model:

Copy the permanent ligation of rats with bilateral arteria carotis communis (2-VO) model, concrete operations are as follows: preoperative 12 h fast of rat are not intake, after 10% chloral hydrate anesthesia, neck preserved skin, as median incision about 1 centimeter, blunt separation subdermal tissue and muscle, be separated bilateral common carotid arteries, No. 1 dual ligation of surgical thread, avoids damage neck sympathetic nerve and vagus nerve.In experimentation, keep autonomous respiration, anus temperature 37 ± 0.5 DEG C, sham operated rats animal surgery process prepares group with model, but not ligation arteria carotis communis.

3, the transplanting of rear MSC set up by rat brain 2-VO model:

Rear 48h set up by rat brain 2-VO model, and control group intervenes umbilical cord mesenchymal stem cells (CI-1M) through tail vein transplantation icarin; Umbilical cord mesenchymal stem cells (48C-1M group): the normal MSC cell suspension 4 × 10 marked through tail venoclysis DIR ⁶/ 0.4ml; Icarin intervenes umbilical cord mesenchymal stem cells (48I-1M) group: the dosing MSC cell suspension 4 × 10 marked through tail venoclysis PKH26 ⁶/ 0.4 ml.According to rat brain stereotaxic atlas, the hippocampus elements of a fix are: ML:-1.62mm, AP:-2.59mm, DV:-3.11mm, marking transfer point, and being drilled in grafting of bone of skull point place with miniature electric, to bore a diameter be 0.5mm aperture.Be fixed on by microsyringe during injection on the directed support of stereotaxic instrument, injected dose is every rat 5 μ l, and inject time is 3min, let the acupuncture needle remain at a certain point 3min, progressively extracts syringe, lasts 3min.After skin suture, abdominal injection gentamicin, every 0.2mL.

4, Testing index:

The impact that transplanted cells is expressed 2-VO rat model hippocampus Nestin, Map2

Within after transplanting the 28th day, collect Ge Zu rat cerebral tissue, be separated hippocampus, the change expressed by Q-PCR investigation hippocampus MAP-2, NESTIN, primer sequence is in table 9.Extract total serum IgE after being separated hippocampus, ultraviolet spectrophotometer measures total rna concentration and purity, saves backup in-80 DEG C.Pcr amplification is carried out with reverse transcription gained cDNA.RT-PCR is with reference to the setting of SYBR Green specification sheets: denaturation 95 DEG C of 15min, sex change 94 DEG C of 30 s, 55 DEG C of 30s, 40 circulations; Reaction terminates rear instrument and automatically generates Ct value and melting curve figure.

Relative quantification calculative strategy is sketched:

This experiment adopt be Δ Δ CT method to carry out relative quantification, method of calculation are as follows:

(1) experimentally measured each gene respectively repeats pipe CT value to calculate this gene mean CT-number.

(2) then deduct the mean CT-number of internal reference gene (being GAPDH in this experiment) with testing gene mean CT-number, obtain the CT value after correcting, i.e. Δ CT.

(3) deduct the correcting CT value of the corresponding gene of authentic specimen by the correcting CT value of each testing gene of laboratory sample, obtain comparative result, i.e. Δ Δ CT.

(4) suppose that the pcr amplification efficiency of each icp gene is consistent and wait with 1 time, the multiple proportion of expression amount in the expression amount of this gene in laboratory sample and authentic specimen can be represented with 2-Δ Δ CT.

Table 9 Q-PCR primer

5, experimental result:

As illustrated in figs. 29-30, Q-PCR result shows, and compare with control group, after hUCMSCs vein transplantation 28d, MSC group and MSC+I group all can significantly improve hippocampus Nestin, MAP-2 genetic expression, but there was no significant difference between group.

Visible, for mouse AKI model, after hUCMSCs vein transplantation 3d and 7d, MSC group and MSC+I group blood BUN and CRE comparatively model group all decrease, and MSC+I group CRE comparatively model group have significance to reduce; Small animal living body imaging results shows, and MSC group and MSC+I group nephridial tissue all detect the MSC signal of mark, and MSC+I group red fluorescent is obviously better than MSC group; Transplant the display of 3d and 7d, HE result, compared with model group, MSC group and MSC+I group all can improve injury of the kidney, and MSC+I group better effects if; Immunofluorescence results display MSC group and MSC+I group all have transplanting MSC to break up to renal cells.

For mouse ALI model, after hUCMSCs vein transplantation 3d and 7d, MSC group and MSC+I group compared with model group and normal group, the horizontal no significant difference of blood ALT and AST; Transplanted cells 3d, compared with model group, MSC group and MSC+I group all can reduce liver coefficient, but have no notable difference between MSC group and MSC+I group; Small animal living body imaging results shows, and MSC group and MSC+I group hepatic tissue all detect the MSC signal of mark, and MSC+I group red fluorescent is obviously better than MSC group; Transplant the display of 3d and 7d, HE result, compared with model group, MSC group and MSC+I group all can improve liver injury; Immunofluorescence results shows, and MSC group and MSC+I group all have transplants MSC to hepatocyte differentiation.

For 2-VO rat model, after hUCMSCs vein transplantation 28d, MSC group and MSC+I group all can significantly improve hippocampus Nestin, MAP-2 genetic expression, but there was no significant difference between group.Result shows, and MSC group and MSC+I group all can improve animal pattern differentiating into nerve cells.

Embodiment 4

The molecular biological mechanism DNA methylation investigating the effect of kidney-nourishing tcm drug activeconstituents based on epigenetics modified mechanism detects:

Umbilical cord mesenchymal stem cells was passaged to for the 4th generation, external evoked one week of icarin 100umol/L, extract DNA, with sodium bisulfite moditied processing genomic dna, allly there is not methylated cytosine(Cyt) (C) to be converted into uridylic (U) methylated cytosine(Cyt) then constant.Genomic dna is after sulfiting, and design BSP primer amplification object fragment, now uridylic (U) is all converted into thymus pyrimidine (T), finally carries out order-checking to PCR primer and just can judge whether CpG site methylates.Carry out the detection of BSP gene methylation.

As shown in Figure 31-32, experimental result shows, adopt the ICA of 100umol/L concentration to make umbilical cord mesenchymal stem cells 1 week, sodium bisulfite sequencing result shows, after ICA effect, and versatility regulatory gene Nanog demethylation change there was no significant difference; The change of versatility regulatory gene Oct-4 demethylation has significant difference, and prompting ICA regulation and control expect that mescenchymal stem cell versatility may methylate relevant with Oct-4 promoter region.

Embodiment 5

Acetylation of histone detects:

Umbilical cord mesenchymal stem cells was passaged to for the 4th generation, and the external evoked cell of icarin 100umol/L, arranges blank group simultaneously, collecting cell after acting on a week.Adopt chromatin immune coprecipitation method, detect the change of two gene promoter region histone modifications

As shown in figure 33; Oct-4 detected result shows; relative to blank group; the ICA of 100umol/L concentration is adopted to make umbilical cord mesenchymal stem cells after 1 week; the Oct-4 DNA of histone H 3 acetylize antibodies measures and obviously increases (P<0.05); result prompting ICA can significantly improve umbilical cord mesenchymal stem cells Oct-4 gene promoter histone H 3 Acetylation Level, and then improves Oct-4 genetic expression.

Visible, kidney-nourishing tcm drug icarin strengthens the expression of umbilical cord mesenchymal stem cells versatility regulatory gene Oct-4 after intervening umbilical cord mesenchymal stem cells, and by showing that icarin 100umol/L effect umbilical cord mesenchymal stem cells can strengthen it to triploblastica histocyte differentiation capability after one week with experiment in vitro in body, further clear and definite kidney-nourishing tcm drug icarin can strengthen the ability of stem cell versatility.By the research of epigenetics modified mechanism, specify that icarin is to the sex molecular biological mechanism of umbilical cord mesenchymal stem cells multipotency.

In sum, the present invention tests by described in embodiment, first kidney-nourishing tcm drug activeconstituents is studied on the impact of umbilical cord mesenchymal stem cells to triploblastica histocyte differentiation capability, the ability that the activeconstituents that external investigation filters out breaks up to triploblastica histocyte hUMSCs, experiment point differentiation-inducing group, icarin (ICA) group, inductor+ICA group, and blank group.Respectively to myocardial cell, neurocyte and the differentiation of islet cells direction, specify that the external ability had to triploblastica cytodifferentiation of hUMSCs.Further, experiment in vivo is implemented on the basis of testing in vitro, by setting up acute liver damage, injury of the kidney, cerebral ischemic model, transplant ICA in body and intervene rear hUMSCs, having investigated kidney-nourishing tcm drug activeconstituents ICA induces rear hUMSCs to transplant Acute Hepatic, the impact of renal failure and the every function criterion of cerebral ischemic model and the impact on transplanted cells differentiation capability in body, demonstrate and intervene hUMSCs by external icarin, after its versatility improves, contribute to hUMSCs in vivo to the histiocytic differentiation of triploblastica, confirm kidney-nourishing tcm drug activeconstituents icarin breaks up versatility impact on hUMSCs further.

Above-mentioned detailed description of the novelty teabag of this icarin being carried out with reference to embodiment; illustrative instead of determinate; several embodiments can be listed according to institute's limited range; therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention.

Claims

1. icarin is strengthening the purposes in stem cell versatility.

2. the purposes of icarin according to claim 1, is characterized in that: by icarin process stem cell, improves stem cell versatility, then transplants stem cell to animal.

3. the purposes of icarin according to claim 1, is characterized in that: be applied to the nutrient solution treating transplanted cells when inducing culture is used for the treatment of acute liver damage or cerebral ischemia.

4. the purposes of icarin according to claim 3, it is characterized in that: the method for described inducing culture cell is: umbilical cord mesenchymal stem cells cultivates 7 days in the perfect medium containing 100 μMs of icarin, within every 3 days, change liquid once, culture condition is 37 DEG C, 5%CO ₂, obtain the stem cell that versatility strengthens, described perfect medium is DMEM-F12 substratum+20%FBS+1%LG+1%PS+10ng/mlEGF+10ng/mlbFGF.