CN101709291A

CN101709291A - Method for in-vitro liver cell differentiation of human marrow mesenchyme stem cells based on VPA inducement

Info

Publication number: CN101709291A
Application number: CN200910155105A
Authority: CN
Inventors: 董学君; 邵健忠; 项黎新; 张卉; 杨超
Original assignee: Shaoxing Peoples Hospital
Current assignee: Shaoxing Peoples Hospital
Priority date: 2009-12-03
Filing date: 2009-12-03
Publication date: 2010-05-19

Abstract

The invention discloses a novel method for the in-vitro liver cell differentiation of human marrow mesenchyme stem cells based on VPA inducement. The human marrow is used as a raw material, the mesenchyme stem cells of the human marrow are separated, a fibroblast growth factor 4, a hepatocyte growth factor, tumour suppressing essence and dexamethasone are sequentially added after the VPA processing of histone ab-acetylase suppressor valproic acid, the liver cells of the human marrow are induced to be differentiated, and a class of polygonal liver cell type cells which have the biological functions of the liver cells of synthesis glycogen, urea, secretion albumin and the like and have double cores are obtained. The method enhances the efficiency of the liver cell differentiation of the human marrow mesenchyme stem cells greatly, provides a novel technology and opens up a novel approach for developing and applying the human marrow mesenchyme stem cells to the cellular transplantation treatment of liver diseases, hepatic tissue engineering and the like and also provides a novel research model for the research of liver cell differentiation development molecular mechanisms comprising a surface appearance genetic mechanism and the like.

Description

A kind of based on the hepatocellular method of VPA inductive human marrow mesenchymal stem cell vitro differentiation

Technical field

The invention belongs to biomedical sector; it is a kind of based on NSC 630176 valproic acid (valproic acid to relate to discovery; VPA) inducing human mesenchymal stem cells (humanbone marrow mesenchymal stem cells, the hBMMSCs) technology of external leap differentiated hepatocellular.

Background technology

Stem cell (Stem cell) is the cell that a class has self and multidirectional differentiation potential, according to the difference of its etap, is divided into embryonic stem cell and adult stem cell.Adult stem cell extensively is present in tissue or organs such as marrow, nerve, muscle, epidermis, intestines, liver.Traditional view is thought adult stem cell only to its types of organization of settling down differentiation, and can not be divided into the cell of other types of organizations.But Recent study shows that adult stem cell can be divided into its place and organize other different types of organization's cells under certain condition, and this is called as plasticity of adult stem cells.The discovery of this potential of adult stem cell, widened its application prospect in the clinical medicine treatment greatly, as leap differentiation potential in view of adult stem cell, utilize adult stem cell directional induction differentiation technique, the adult stem cell that derives from different tissues can be divided into the cell of required tissue, as utilize hemopoietic stem cell, can not only rebuild patient's hematopoiesis and immunologic function, can also in patient's body, be divided into various sophisticated non-hemopoietic tissue cells, as the myocardial cell, liver cell, epithelial cell, Skeletal Muscle Cell etc., these cells can organically be incorporated in corresponding tissue and the organ, repair or replace patient's loss of function or impaired tissue and organ.

Many intractable hepatopathys such as chronic hepatitis, liver cirrhosis, portal hypertension disease, primary hepatocarcinoma, heredity and metabolic hepatic diseases have become the thorny problem that the whole world faces jointly, and China every year is because of about 500,000 people of patient of hepatopathy death.Current, mainly depend on orthotopic liver transplantation for the treatment of liver failure (as hepatic necrosis, the liver cirrhosis etc.) heredopathia relevant, but problems such as donor livers source wretched insufficiency and transplant rejection have limited extensively carrying out of this treatment means with liver.Hepatocyte transplantation and bioartificial liver are as the supplementary mode of liver transplantation, can partly alleviate the problems referred to above, but it is short that its derived cell equally also is faced with survival time, particularly along with the prolongation of incubation time, problems such as liver function reduces gradually, therefore, seek suitable liver cell source and become a very urgent subject.Be divided into the effective way that liver cell addresses this problem beyond doubt and utilize adult stem cell to cross over, so the research of this respect also becomes one of research focus of current stem cell and hepatopathy cell therapy.

Usually BMMSCs can be divided into scleroblast, adipocyte and chondrocyte, but studies have shown that much that in recent years BMMSCs in vivo or the potential of external oriented various kinds of cell system differentiation, for example epidermal-like cell, neurocyte and liver cell.These characteristics of BMMSCs for them when using in the future, can avoid a lot of obstacles, for example utilize from the cell of body BMMSCs directed differentiation and transplant, can avoid the risk of Bioethics problem and immunological rejection, make it well to select as organizational project and cellular transplantation therapy one.Yet setting up effective BMMSCs is crucial to liver cell vitro differentiation scheme.In recent years; the investigator utilizes experimental animal models such as rat and mouse; BMMSCs vitro differentiation liver cell has been carried out more research; and some induction technology have tentatively been set up; as utilize mouse liver injury organization condition nutrient solution and the hepatocellular technology of tire hepatic tissue conditioned medium inducing mouse BMMSCs vitro differentiation; utilize different cytokines (as SCF; HGF; EGF and FGF-4 etc.) make up and induce differentiation technique etc.; but the research about people BMMSCs differentiated hepatocellular is still rare; and research work concentrates on mostly and sets up different combination of cytokines and induce differentiation, and Shang Weijian utilizes NSC 630176 VPA to induce the technical system of its differentiated hepatocellular.

Summary of the invention

The technical problem to be solved in the present invention is to set up a kind ofly can improve the hepatocellular new technology of human marrow mesenchymal stem cell (hBMMSCs) vitro differentiation by a relatively large margin.In order to solve the problems of the technologies described above, to the invention provides a kind of regulation and control acetylation of histone epigenetic modification that passes through and promote liver cell differentiation regulate gene expression, thereby realize the technology of liver cell differentiation efficiency.Wherein the regulation and control of Mechanisms of Histone Acetylation Modification are passed through to use histon deacetylase (HDAC) (Histonedeacetylases; HDACs) inhibitor VPA realizes; improvement as the inventive method; after using VPA pre-treatment hBMMSCs; be combined into fibroblast growth factor 4 (FGF4), pHGF (HGF), tumour inhibitor (OSM) cytokine and induce, to promote hBMMSCs to hepatocellular early stage differentiation and later stage maturation.

In order to solve the problems of the technologies described above, the present invention realizes by the following method:

A kind of based on the hepatocellular method of VPA inductive human marrow mesenchymal stem cell vitro differentiation, this method is that the marrow with the people is raw material, by separating its mescenchymal stem cell, after NSC 630176 VPA handles, add FGF4, HGF and OSM and Dex successively, induce it to break up to liver cell; Comprise the steps:

(1) separation of mesenchymal stem cells MSCs and preparation;

(2) carry out the VPA pre-treatment after, different cytokines array mode inducing culture cell is to the differentiation of inducing of skeletonization, fat and cartilage direction;

(3) evaluation of differentiated hepatocellular.

Separation and preparation in the described step (1) comprise the steps:

A) obtain marrow from the adult healthy volunteer;

B) with density gradient centrifugation separation mononuclearcell wherein;

C) with the mescenchymal stem cell in the difference growth adherent method purification of individual karyocyte.

Raw material in the described step a) is taken from adult healthy volunteer's marrow, and adds the anticoagulant heparin agent, and the anticoagulant heparin agent dose is 10.0～12.5IU/ml blood.

Mononuclearcell obtains by following method in the described step b): with the artificial sera (PBS of phosphate buffered, pH7.0) the marrow washing that will from step a), obtain 2 times, and marrow is diluted to cell suspension, add to human lymphocyte parting liquid (Ficoll-Hypaque, the upper strata of D=1.077 ± 0.002g/ml), behind centrifugal 25 minutes of the room temperature 2200rpm/min, get middle white cellular layer (mononuclearcell layer), add the basic culture solution washed cell, the 1000rpm low-speed centrifugal is 6～7 minutes then, abandon supernatant, obtain people's BMNC.

Described step c) is after will suspending again with the basic culture solution that contains 10%FBS through the BMNC that step b) obtains, to press 4 * 10 ⁴/ cm ²Density is inoculated in the disposable plastic culturing bottle, puts 37 ℃, 5%CO ₂Cultivate in the incubator, remove nutrient solution behind the 48h, with twice of PBS flushing, discard not adherent cell, add fresh basic culture solution and continue to cultivate, to cover the culturing bottle floorage about 70%～90% the time when primary cultured cell, with 0.25% tryptic digestion collecting cell, be seeded in the orifice plate by limiting dilution assay, be the stem cell (P1) of the first-generation this moment; Changed fresh basic culture solution in per 3 days, and chose form homogeneous, well-grown cell, use the trysinization collecting cell, the cultivation of proceeding to go down to posterity obtains containing the hBMMSCs of single purifying.It is the third generation to the stem cell in seven generations (P3～P7) that this experiment middle and later periods is used for the inductive stem cell.

Inducing culture cell in the described step (2) is to add inductor in the basic culture solution of the human marrow mesenchymal stem cell that contains single purifying that obtain after will handling through step (1); The nutrient solution that inductor adopts NSC 630176 VPA and contains cytokine; At first add the VPA pre-treatment after 72 hours, removal contains the nutrient solution of valproic acid, add the nutrient solution that contains cytokine, described cytokine is respectively FGF4, HGF, OSM and Dex, and this addition sequence is similar to the secretion order of cytokine in the liver ontogenetic process; The nutrient solution that wherein contains cytokine is meant all first proportionally the joining in the basic medium of cytokine is obtained; Changed in per three days and state nutrient solution, inducing culture to 7 day respectively, 14 days and 21 days, obtain liver cell based on VPA inductive hBMMSCs vitro differentiation, carry out the evaluation that liver cell breaks up then.

The add-on of described cytokine is respectively: 5mM VPA, (mM refers to mmol/l) 20ng/ml FGF4,30ng/ml HGF, 20ng/ml OSM and 40 μ g/ml Dex.

The evaluation of the liver cell differentiation in the described step (3) comprises: the immunofluorescence of morphological observation, liver cell specific gene and protein expression (RT-PCR) analysis, the detection of cell glycogen synthesis capability, urea synthesis Function detection, function of albumin secretion detection, the active detection of Cytochrome P450, cell induction histone H 3 and H4 detects and the specific proteins marking (westernblot) detects.

Acetylation of histone be find the earliest with transcribe one of relevant histone modification mode.(Histoneacetyltransferases, HATs) (Histonedeacetylases is keeping running balance under synergy HDACs) to the acetylize state of histone end with the histone deacetylase enzyme at histone acetyltransferase.HATs can transfer to the ethanoyl of acetyl-CoA on the lysine residue of histone, make its epsilon-amino group acetylize, thereby make chromosome structure loose, help transcription factor, RNA polymerase and transcription complex etc. and enter and its corresponding, promote to transcribe in conjunction with DNA site (promotor and enhanser etc.) combination; The then removable ethanoyl of HDACs makes dna methylase inhibitor, forms the high-grade chromosome structure, and suppresses the transcription complex assembling, thereby suppresses to transcribe.The active balancing control of HATs and HDACs the acetylize level of nucleosome core histone, and then influences the transcriptional activity of genes involved.The experiment that changes by structure and expression level to HATs in the kinds of tumor cells and HDACs confirms that acetylation of histone and deacetylation have critical function in cell proliferation, differentiation and apoptosis.At present existing multiple HDACs inhibitor is used as carcinostatic agent and carries out clinical and experimental study, common HDACs inhibitor have valproic acid (valproic acid, VPA), butyric acid (butyrate acid) and trichomycin A (Trichostatin A, TSA) etc.The HDACs inhibitor has selectively acting for normal cell, though can change gene expression of cells, can not cause Normocellular apoptosis.BMMSCs is a kind of normal cell, can cause the variation of genetic expression under the effect of VPA, but can't cell death inducing.

The present invention utilizes above-mentioned principle, adopt VPA to induce hBMMSCs, and induce in conjunction with FGF4, HGF and OSM combination, successfully set up a kind of technical system of the new external leap differentiated hepatocellular of hBMMSCs, significantly improved the vitro differentiation efficient of human liver cell, for the hBMMSCs Application and Development in the cellular transplantation therapy of hepatic diseases and carry out liver tissue engineering etc. and new technology is provided and has opened up new approach.

The present invention has following advantage with respect to prior art;

(1) present technique induces hBMMSCs to cross over differentiation to liver cell with 5mM VPA in conjunction with the IMDM basic culture solution of three kinds of cytokines (20ng/ml FGF-4,30ng/ml HGF, 20ng/ml OSM) and Dex (40 μ g/ml), the formation of this technology is compared with the numerous combination of cytokines system of inducing that adopts usually at present, and is simple relatively.

(2) induce effect obvious, pass through morphological observation, the liver cell expression of specific gene is analyzed, the immunofluorescence of liver cell differential protein detects, PAS reaction detection cell glycogen is synthetic, technology and indexs such as Cytochrome P450 determination of activity and urea synthesis ability, identification of cell differentiation situation and efficient thereof, and experimental group and control group compared, the result shows, the differentiation efficiency of experimental group cell is significantly higher than control group, with secretion albumin (ALB) and urea synthesis is functional parameter, demonstration improves 50%～62% through the differentiation rate of the experimental group cell that VPA handles, the index that is expressed as with the early stage differentiation gene AFP of liver cell, show between the differentiation phase of experimental group cell than an about in advance week of control group, show that system of the present invention can significantly promote hBMMSCs to hepatocellular external leap differentiation efficiency, shorten the required time of cytodifferentiation.

(3) because relating to, the mesenchymal stem cells MSCs differentiated hepatocellular strides the germinal layer differentiation, this is a new proposition of developmental biology research, VPA is a kind of epigenetic modification agent simultaneously, therefore, the present invention not only provides the more efficiently hepatocellular method of obtaining for hBMMSCs is applied to the hepatopathy cellular transplantation therapy, also provides new research model for the research of liver cell differentiation and development molecular mechanism (comprising epigenetic mechanism etc.).

Description of drawings

Table 1 is a cDNA reverse transcription synthetic system table of the present invention;

Table 2 is pcr amplification primer tables of the present invention;

Table 3 is PCR reaction system tables of the present invention;

Fig. 1 is the coloration result evaluation figure of the isolating human marrow mesenchymal stem cell of the present invention to scleroblast, chondrocyte and adipocyte differentiation;

Fig. 2 is that the present invention induces the experimental system schema of human mesenchymal stem cell to the liver cell differentiation;

Fig. 3 is the morphologic observation of inventor's mesenchymal stem cells MSCs in liver cell atomization figure as a result;

Fig. 4 is that inventor's mesenchymal stem cells MSCs liver in the liver cell atomization is the analysis chart of expression of specific gene;

Fig. 5 is that inventor's mesenchymal stem cells MSCs liver in the liver cell atomization is the analysis chart that specific proteins is expressed;

Fig. 6 is the expression figure that liver cell breaks up mark AFP in early days in the ELISA detection cell differentiation procedure of the present invention;

Fig. 7 is the detected result figure of function of albumin secretion of the present invention;

Fig. 8 is urea synthesis Function detection of the present invention figure as a result;

Fig. 9 is the analytical results figure of chromatin histone (H3 and H4) acetylation modification level before and after VPA of the present invention handles.

Embodiment

Embodiment 1

The separation of human marrow mesenchymal stem cell and cultivation

At first from people's marrow sample, separate mononuclearcell, to take from adult healthy volunteer's marrow, with one to twice of phosphate buffered saline buffer (PBS) washing, and marrow is diluted to cell suspension, add to human lymphocyte parting liquid upper strata (Ficoll-Hypaque, D=1.077 ± 0.002g/ml) gently; Behind centrifugal 25 minutes of the room temperature 2200rpm/min,, get middle white cellular layer (mononuclearcell layer) with upper strata PBS sucking-off; Add basic culture solution and (contain 10% foetal calf serum FBS, the low sugar IMDM of 100mg/ml Streptomycin sulphate 100U/ml penicillin) washed cell, 1000rpm/min low-speed centrifugal 6～7min then, remove nutrient solution, and cell dilution is become certain density suspension with this nutrient solution, be inoculated in the culturing bottle, put 37 ℃, the 5%CO2 incubator is cultivated, change nutrient solution behind the 48h first, discard not adherent cell, can observe a small amount of short fusiformis, stellate cell disperses adherent growth, the cell colony of visible radial arrangement behind continuation cultivation 24～48h, stretch out different in size, the projection of thickness inequality, karyon is big, kernel is clear, continue to be cultured to 80%～90% degree of converging, be the whirlpool shape, digest collecting cell with pancreatin/EDTA, the low density inoculation culture, passage cell is easy to adherent than primary cell, cell stretches more, form is homogeneous more, treats that cell grows to 75% degree of converging, the cultivation of can going down to posterity, by the separation and Culture of cell, obtain the single fibrous cell of form.

The evaluation of human marrow mesenchymal stem cell differentiation capability

Adopt differentiation potential sexual function evaluation assessment to identify isolating human marrow mesenchymal stem cell (hBRMSCs).Concrete grammar is: the cell that will be cultured to for the 3rd generation carries out the differentiation of inducing to skeletonization, fat and cartilage direction.When cell grows to 80% left and right sides density, add different induced liquids.Inducing cell is to osteoblast differentiation, adds 10mM sodium (β-glycerophosphate), 50mg/ml xitix (ascorbicacid) and 10 in basic culture solution ^-7(dexamethasone Dex), cultivated for three weeks to the M dexamethasone, changed liquid once in per three days.Detect osteoblastic differentiation (shown in Figure 1A, B, C) with alkaline phosphatase, sodium alizarinsulfonate and Von Kossa dyeing process; Inducing cell breaks up to adipocyte, adds 50 μ g/ml Regular Insulin (insulin) and 10 in basic culture solution ^-7M Dex, cultivate 2-4 week, changed a nutrient solution in per three days, observe adipocyte (shown in Fig. 1 E) with phase microscope, simultaneously cell is washed twice with PBS, fix 10 minutes with 4% Paraformaldehyde 96 room temperature, the distilled water rinsing, added oil red 0 (6 part of 0.6% oil red 0 that is dissolved in Virahol adds 4 parts of water) room temperature reaction 1 hour, rinsing, phase microscope is observed, (shown in Fig. 1 F) takes pictures; Inducing cell breaks up to the chondrocyte, in basic culture solution, add 10ng/ml transforming growth factor-beta (Transforming growth factor beta, TGF-β), 50 μ g/ml vitamins Cs, changed a nutrient solution in per three days, cultivated for three weeks, noble cells dyes with alcian blue, and phase microscope is observed coloration result (shown in Fig. 1 D).Fig. 1 is that the differentiation function of isolating hBMMSCs is identified, shows that it has the potential that typically breaks up to skeletonization, cartilage and adipocyte.A, B, C are respectively alkaline phosphatase, sodium alizarinsulfonate and von-kossa dyeing and detect osteoblast differentiation; D is that alcian blue dyeing detects chondrocyte's differentiation; E is the adipocyte morphologic observation of differentiation; F be the oil red 0 dyeing formation that detects fat and drip (A, B, C, D 50 *, E 200 *, F 400 *).Above result proves that we isolating hBMMSCs can have multiple differentiation potential to skeletonization, fat and chondrocyte's differentiation.

Human marrow mesenchymal stem cell is to liver cell induced differentiation and evaluation

Choose and be cultured to eugonic hBMMSCs cell of P3 generation, and it is divided into two groups of A, B, the A group is as experimental group, cell usefulness was contained the basic medium pre-treatment of 5mM VPA after 3 days, adopt the method for fractional steps system of inducing as shown in Figure 2 further to induce, A is for using VPA inductive experimental group, and B is without VPA inductive control group.

Concrete operations are to add the basic culture solution that contains FGF4 (20ng/ml) earlier to induce three days, change the basic culture solution that contains HGF (30ng/ml) then, induced three days, changing the basic culture solution of the hybrid cytokine that contains HGF (30ng/ml), OSM (20ng/ml) and Dex (40 μ g/ml) again cultivates, changed above-mentioned nutrient solution in per three days, every day is the observation of cell metamorphosis under phase microscope, breaks up detection in 7,14 and 21 days at inducing culture to the respectively.

The evaluation of liver cell differentiation comprises: morphological observation, liver cell expression of specific gene are analyzed, the immunofluorescence technique of liver cell differential protein detects, Periodic acid-Schiff (PAS) reaction detection cell glycogen is synthetic, Cytochrome P450 is active and the urea synthesis Function detection.

(1) morphological observation: in the cell differentiation procedure, day by day the variation of observation of cell form under phase microscope, and be induced to the 7th day (A as shown in Figure 3 and B, 100 *), 14 days (C as shown in Figure 3 and D, 100 *) and 21 days (E as shown in Figure 3 and F, 100 *) time Taking Pictures recording, the relatively difference on two groups of cellular fories.

The VPA untreated fish group is three groups of A, C, E; The VPA treatment group is three groups of B, D, F.A be normal cultivate without the pretreated hBMMSCs of VPA, its form presents fusiformis or fusiform, monokaryon substantially; B is the cellular form after VPA handles 72h, and visible fusiformis or fusiform hBMMSCs begin to break up to Polygons; C, D and E, F are respectively the cellular form when continue adding

cytokine induction

14 and 21 days, show cellular form to epithelium sample or Polygons transform, multinuclear appears in the part cell, and the cell of VPA treatment group is more obvious to the variation of epithelium sample and Polygons form.

(2) RT-PCR of liver cell specific gene expression detects with Real-time PCR: the cell of getting different induction time points (the 7th and 21 day), with Trizol test kit (TaKaRa company) extracted total RNA, become the genes such as β-actin, AFP, HNF3 β and ALB that increase respectively behind the cDNA with reverse transcription test kit (TaKaRa company) reverse transcription; Synthetic use RT test kit TaKaRa RNA PCR Kit (AMV) Ver.3.0 of cDNA first chain carries out, see Table 1, the PCR reaction system sees Table 3, and concrete reaction conditions is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 54～60 ℃ of annealing 30s, 72 ℃ are extended 30s, 30～40 circulations, and 72 ℃ are extended 10min, annealing temperature and cycle number are selected according to the particular case of primer, and the PCR primer sees Table 2.Get 3 μ l amplified productions after reaction is finished and add 1 μ l bromjophenol blue indicator, the agarose with 1.2% carries out gel electrophoresis.After electrophoresis finished, (Eastman Kodak Company USA) analyzed with Kodak GEL LOGIC 200 gel imaging analysis systems.The expression amount of fluorescence quantitative PCR detection AFP and ALB: select for use

Premix Ex Taq ^TMKit (TaKaRa) test kit carries out on Mastercycler ep realplex (Eppendorf) instrument.10 μ l reaction systems are all used in all Real-Time PCR reaction, use simultaneously that β-the actin gene is as confidential reference items, and experiment condition is as follows: (1) two-step approach circulation 40 times, 95 ℃ of 30s, 60 ℃ of 20s; (2) Melting curve analyzes, 95 ℃ of 5s, 65 ℃ of 15s, 95 ℃ of 15s; (3) cooling, 40 ℃ of 30s; Each sample cDNA carries out homogenization with internal control gene.Fig. 4 is that human mesenchymal stem cell liver in the liver cell atomization is the analysis of expression of specific gene, and A represents among the figure: control group (VPA when inducing 7 days and 21 days ^-) and experimental group (VPA ⁺) AFP, HNF3 and ALB expression of gene in the cell, the result shows that through the experimental group cell that VPA handles, its level of expressing AFP, HNF3 β and ALB obviously strengthens, and the time of expressing is in advance, shows that VPA has promoted hepatocellular differentiation.B and C represent to show among the figure: quantitative fluorescent PCR detects the expression amount of ALB and AFP when inducing 7 days, 14 days and 21 days respectively, and the result shows that the VPA pretreated group compares with the VPA untreated fish group, and the expression amount of ALB and AFP obviously raises.(P＜0.01, n=5) same, (p＜0.01, n=5), HepG2 is the hepatic cell line positive control to the remarkable control group of expression amount of ALB among the figure when experimental group the 14th and 21 days wherein to be significantly higher than control group through the expression amount of experimental group AFP in the time of the 7th day.

(3) immunofluorescence dyeing: get the cell of different induction time points, immunofluorescence staining carries out mark routinely, and concrete steps are as follows: inhale the nutrient solution that goes in the orifice plate to be measured, with PBS rinsing cell 3 times, each 5min adds 4% Paraformaldehyde 96 stationary liquid, and room temperature is 15min fixedly; Paraformaldehyde 96 is removed in suction, adds-20 ℃ of pre-cold methanols and handles 20min, removes methyl alcohol, and is air-dry; Add 5% normal goats serum confining liquid, act on 20min under the room temperature; 1h (or 4 ℃ wet box in overnight incubation) is hatched in one anti-(goat-anti people ALB antibody and the anti-people AFP of rabbit antibody, the Abcam company) that adds dilution in 1: 100～1: 1000 in 37 ℃ of wet boxes; Suction goes one to resist, PBS rinsing 3 times, each 5min; Fluorescein-labelled two anti-(anti-sheep IgG of the rabbit of FITC or TRITC mark and the mouse-anti rabbit iggs that add dilution in 1: 100～1: 1000, Peprotech company), hatch 30～45min in 37 ℃ of wet boxes, PBS rinsing 3 times, each 5min, (A as shown in Figure 5, B, C and D, 100 *) observed down and taken pictures to laser confocal microscope.

(4) the glycogen complex functionality is measured: the synthetic and memory function of the hepatocellular glycogen of PAS reaction detection.Choose the cell of inducing 21 days, remove induced liquid, wash 2 times with DPBS, 95% alcohol fixation cell 10min, distilled water flushing 1min adds 1% periodic acid aqueous solution effect 15min, behind the distilled water flushing 1min again, add Schiff reagent effect 30min, rocked every several minutes the centre, and distilled water flushing 1min is used in sulfurous acid washing 3 times again, microscopy under the phase microscope, (E as shown in Figure 5 and F, 100 *) takes pictures.

(5) Cytochrome P450 determination of activity: choose the cell of inducing 21 days, remove induced liquid, adding final concentration in normal nutrient solution is 5 μ M phenylethyl barbituric acids, induce 24h in the incubator, sopped up inducing culture liquid in second day, DPBS washes once, replacing is added with the nutrient solution of 5 μ Methoxyresorufin, after hatching 2～3h, discard nutrient solution, DPBS washes 3 times, adopts Carl Zeiss laser confocal scanning microscope to observe photographic analysis (355nm excitation and 581nm emission) (G as shown in Figure 5 and H, 100 *).

Fig. 5 is that human mesenchymal stem cell liver in the liver cell atomization is analysis and the functional examination figure that specific proteins is expressed, and A, B and C, D are respectively when inducing 7 days and 21 days AFP and the proteic immunofluorescence dyeing of ALB in the control group and experimental group cell among the figure; E, F are the PAS staining analysis of inducing glycogen synthesis capability in 21 o'clock control groups and the experimental group cell; G, H be control group (VPA when inducing 21 days ^-) and experimental group (VPA ⁺) Cytochrome P450 activation analysis in the cell.The result shows that through the cell that VPA handles, its level of expressing AFP and ALB obviously strengthens, and PAS positive cells quantity increases and the PAS increased response simultaneously, and the Cytochrome P450 increased activity shows that VPA has significantly promoted hepatocellular differentiation.

(6) ELISA detects the expression amount of alpha-fetoprotein AFP in the cell differentiation procedure: choose different induction time points (0,7,14 and 21 days) cell (establish the blank inductive hMSSCs of PBS as negative control group, VPA handles induces group and normally induces group without what VPA handled), remove induced liquid, clean cell 3 times with PBS, adding low serum nutrient solution (2% foetal calf serum) cultivated 24 hours, collect nutrient solution, 10000rpm removed impurity in centrifugal 15 minutes, collect supernatant, automatic clinical chemistry analyzer detects AFP content (as shown in Figure 6), Fig. 6 is the detection figure of AFP secreting function, shown in the figure: through experimental group in the time of the 7th day, the expression amount of its AFP increases (P＜0.01 than cellular control unit is remarkable, n=5), experiment is established mescenchymal stem cell without any processing as negative control group.

(7) function of albumin secretion is measured: the cell (establishing the blank inductive hMMSCs of PBS induces group and normally induce group without what VPA handled as what negative control group, VPA handled) of choosing different induction time points (0,7,14 and 21 day), remove induced liquid, clean cell 3 times with PBS, adding low serum nutrient solution (2% foetal calf serum) cultivated 24 hours, collect nutrient solution, 10000rpm/min removed impurity in centrifugal 15 minutes, collect supernatant, automatic clinical chemistry analyzer detects albumin content (as shown in Figure 7).Fig. 7 is the detection figure of function of albumin secretion, shown in the figure: with compare without the VPA treatment group, the cell of handling through VPA is when inducing 14 days, the expression amount of its ALB begins obvious increase (P＜0.01, n=5), simultaneously with undressed mescenchymal stem cell as negative control group.

(8) urea synthesis is measured: the cell (establishing the blank inductive hMSSCs of PBS induces group and normally induce group without what VPA handled as what negative control group, VPA handled) of choosing different induction time points (0,7,14 and 21 day), remove induced liquid, wash 2 times with PBS, add and contain 5mol/l NH ₄The IMDM nutrient solution of Cl is hatched 24h for 37 ℃.Collect nutrient solution, the centrifugal 10min of 1500rpm/min gets supernatant, and automatic clinical chemistry analyzer and urea detection kit are measured the urea content (as shown in Figure 8) in the nutrient solution supernatant.Fig. 8 is the urea synthesis Function detection, show after the groups of cells that VPA handles is being induced 7 days, its urea synthesis begins to increase than untreated groups of cells with secretory volume, after 14 days, its secretory volume obviously increases (P＜0.01, n=5), experiment is established undressed mescenchymal stem cell as negative control group.

The epigenetic modification analysis of VPA mediation

The epigenetic modification of VPA mediation mainly is that the pair cell acetylation of histone exerts an influence.The mesenchymal stem cells MSCs that is cultured to P3 generation with 5mM VPA pre-treatment 72 hours, is then added combination of cytokines mode in the present embodiment with this.Acetylizad histone is observed by cellular immunofluorescence staining and western blot with anti-acetylated histones H3 and H4 antibody.

(1) cellular immunofluorescence staining: the above-mentioned VPA inductive cell of learning from else's experience, immunofluorescence staining carries out mark routinely, and concrete steps are as follows: inhale the nutrient solution that goes in the orifice plate to be measured, with PBS rinsing cell 3 times, each 5min adds 4% Paraformaldehyde 96 stationary liquid, and room temperature is 15min fixedly; Paraformaldehyde 96 is removed in suction, adds-20 ℃ of pre-cold methanols and handles 20min, removes methyl alcohol, and is air-dry; Add 5% normal goats serum confining liquid, act on 20min under the room temperature; Add one anti-(goat-anti people acetylize H3 and acetylize H4 antibody, Abcam company) of dilution in 1: 100, hatch 1h in 37 ℃ of wet boxes; suction goes one to resist; PBS rinsing 3 times, each 5min adds fluorescein-labeled two of dilution in 1: 100 again and resists (the anti-sheep IgG of the rabbit of FITC or TRITC mark; Peprotech company); hatched 30 minutes PBS rinsing 3 times, each 5min in 37 ℃ of wet boxes; (A as shown in Figure 9,1000 *) observed down and taken pictures to laser confocal microscope.

(2) Western blot analyzes: discrete group albumen at first, will (press 10 with the TEB damping fluid through the cell that trysinization is collected PBS washed twice ⁷/ ml density) suspension cell is put ice bath 10min with the dissolved cell, rocks gently; 4 ℃ of centrifugal 10min abandon supernatant, use the TEB washed cell, add 0.2M HCl (by 4 * 10 ⁷Density) lysing cell, 4 ℃ are spent the night, 4 ℃ of next day are centrifugal 10 minutes, get supernatant liquor, the 15%SDS-PAGE protein isolate, electricity changeed the purpose bar and brings on the wetting nitrocellulose filter of deionized water (NC) film, and the flush away electricity changes damping fluid, with a little ponceau dyeing 10 seconds, observing changes the film effect, with the PBST closing membrane that contains 20%BSA, 4 ℃ are spent the night, discard confining liquid, add anti-H3 and H4 antibody by 1: 2000 and 1: 10000 extent of dilution respectively, 4 ℃ are spent the night, and clean one with PBST and resist, and add two anti-Incubating Solutions by 1: 5000 extent of dilution then, PBST flush away two is anti-, Dropwise 5 00ul left and right sides luminescent solution behind the slightly dried NC film, exposure is 20 seconds in the X-ray folder, through developing, photographic fixing, clean the back and observe (B as shown in Figure 9).

Fig. 9 is the analysis of chromatin histone (H3 and H4) acetylation modification level before and after VPA handles; A figure is that the immunofluorescence of Mechanisms of Histone Acetylation Modification level detects, and wherein (a) is the acetylize dyeing of H3 histone in examining without the cellular control unit that VPA handles; (b) be the acetylize dyeing of H3 histone in 72 hours the nucleus of VPA pre-treatment; (c) be the acetylize dyeing of H4 histone in examining without the cellular control unit that VPA handles; (d) be the acetylize dyeing of H4 histone in 72 hours the nucleus of VPA pre-treatment; The degree of acetylation that can be observed two kinds of histones in the pretreated nucleus of VPA all is significantly increased.B figure is that the Western blot of Mechanisms of Histone Acetylation Modification level detects, and shows that the hybridization signal of H3 and H4 obviously strengthens in the VPA treatment group cell, and the acetylize that shows histone H 3 and H4 is handled the back at VPA significantly to be increased.

Table 1

Table 2

Annotate: AFP (α-fetoprotein); HNF3 β (hepatocyte nuclear factor 3 β, alternatively calledFOXA2); ALB (albumin)

Table 3

Claims

1. one kind based on the hepatocellular method of VPA inductive human marrow mesenchymal stem cell vitro differentiation, it is characterized in that this method is that marrow with the people is raw material, by separating its mescenchymal stem cell, after the NSC 630176 valproic acid is handled, add fibroblast growth factor 4, pHGF, tumour inhibitor and dexamethasone successively, induce it to break up to liver cell; Comprise the steps:

(1) separation of mesenchymal stem cells MSCs and preparation;

(2) carry out the valproic acid pre-treatment after, different cytokines array mode inducing culture cell is to the differentiation of inducing of skeletonization, fat and cartilage direction;

(3) evaluation of differentiated hepatocellular.

2. as claimed in claim 1 a kind of based on the hepatocellular method of VPA inductive human marrow mesenchymal stem cell vitro differentiation, it is characterized in that separation and the preparation in the described step (1) comprises the steps:

A) obtain marrow from the adult healthy volunteer;

B) with density gradient centrifugation separation mononuclearcell wherein;

C) with the mesenchymal stem cells MSCs in the difference growth adherent method purification of individual karyocyte.

As claim 2 state a kind of based on the hepatocellular method of VPA inductive human marrow mesenchymal stem cell vitro differentiation, it is characterized in that the raw material in the described step a) takes from adult healthy volunteer's marrow, and adding the anticoagulant heparin agent, the anticoagulant heparin agent dose is 10.0～12.5IU/ml blood.

As claim 2 state a kind of based on the hepatocellular method of VPA inductive human marrow mesenchymal stem cell vitro differentiation, it is characterized in that mononuclearcell obtains by following method in the described step b): the marrow washing that will from step a), obtain with the artificial sera of phosphate buffered 2 times, and marrow is diluted to cell suspension, add to human lymphocyte parting liquid upper strata, behind centrifugal 25 minutes of the room temperature 2200rpm/min, mononuclearcell layer in the middle of getting, add the basic culture solution washed cell, the 1000rpm low-speed centrifugal is 6～7 minutes then, abandon supernatant, obtain people's BMNC.

As claim 2 state a kind of based on the hepatocellular method of VPA inductive human marrow mesenchymal stem cell vitro differentiation, it is characterized in that described step c) is after will suspending again with the basic culture solution that contains 10%FBS through the BMNC that step b) obtains, to press 4 * 10 ⁴/ cm ²Density is inoculated in the disposable plastic culturing bottle, puts 37 ℃, 5%CO ₂Cultivate in the incubator, remove nutrient solution behind the 48h, with twice of PBS flushing, discard not adherent cell, adding fresh basic culture solution continues to cultivate, to cover the culturing bottle floorage about 70%～90% the time when primary cultured cell, with 0.25% tryptic digestion collecting cell, is seeded in the orifice plate by limiting dilution assay; Changed fresh basic culture solution in per 3 days, and chose form homogeneous, well-grown cell, use the trysinization collecting cell, the cultivation of proceeding to go down to posterity obtains containing the basic culture solution of the human marrow mesenchymal stem cell of single purifying.

As claim 1 state a kind of based on the hepatocellular method of VPA inductive human marrow mesenchymal stem cell vitro differentiation, it is characterized in that inducing culture cell in the described step (2) is to add inductor in the basic culture solution of the human marrow mesenchymal stem cell that contains single purifying that obtain after will handling through step (1); The nutrient solution that inductor adopts the NSC 630176 valproic acid and contains cytokine; At first add the valproic acid pre-treatment after 72 hours, removal contains the nutrient solution of valproic acid, add the nutrient solution that contains cytokine, described cytokine is fibroblast growth factor 4, pHGF, tumour inhibitor and dexamethasone, and the nutrient solution that wherein contains cytokine is meant all first proportionally the joining in the basic medium of cytokine obtained; Changed in per three days and state nutrient solution, inducing culture to 7 day respectively, 14 days and 21 days, obtain liver cell based on VPA inductive human marrow mesenchymal stem cell vitro differentiation, carry out the evaluation that liver cell breaks up then.

As claim 6 state a kind of based on the hepatocellular method of VPA inductive human marrow mesenchymal stem cell vitro differentiation, it is characterized in that the add-on of described inductor is respectively: the 5mM valproic acid; 20ng/ml fibroblast growth factor 4; The 30ng/ml pHGF; 20ng/ml tumour inhibitor and 40 μ g/ml dexamethasone.

As claim 1 state a kind of based on the hepatocellular method of VPA inductive human marrow mesenchymal stem cell vitro differentiation, it is characterized in that the evaluation of the liver cell differentiation in the described step (3) comprises: morphological observation, liver cell specific gene and protein expression analysis, detections of cell glycogen synthesis capability, urea synthesis Function detection, function of albumin secretion detect, Cytochrome P450 actively detects, the immunofluorescence of cell induction histone H 3 and H4 detects and the detection of the specific proteins marking.