Investigation Of Rat Bone Marrow-derived Mesenchymal Stem Cells Differentiate Into Hepatocytes

Mesencymal stem cells is a kind of cells in bone marrow.They have the capable of differentiation into various organize cells and can amplify in vitro.We identified by immunocytochemistry and the method of differentiating into adipocytes ,in order to prove what we have cultured was MSCs.The result was that the isolated cells had the characteristics of MSCs.One of the hotspot of basic investigation is how to supply a kind of seed cell for the patients with liver failure.The aim of hepatocyte transplantation is to repaire organize by supplying active cells.Up on now ,the method for inducing MSCs into hepatocyte is that:1.To differentiate MSCs into hepatocyte by appending some cytokines in L-DMEM.Kang.etal.had differentiated rMSCs into hepatocyte by appending HGFandFGF-4.It was proved that cytokines may play a more important role in differentiation from rat MSCs into hepatocyte.2.Appending serum of hepatopath and so on in medium. Yamazaki etal.had used medium with serum of hepatopath,HGF,dexamethasone to differentiate MSC.After tow weeks it was detected the expression of hepatocyte.So they think that it is a suitable condition to differentiate into heatocyte.3.Cocultured with hepatocyte. Miznguchi.etal.had improved the rate of hepatocyte differentiation by coculturing MSCs and hepatocyte.4.By gene transplantation.We investigate that rMSCs differentiate into hepatocyte with drug.To supply a seed cell for hepatocyte transplantat(HT).[OBJECTIVES]The aim of this study is to research on the method of isolation,purification and culture of rat bone marrow (rMSCs) .We explore the best way to identify the rMSCs and induce the rMSCs into hepatocyte with liver homogenate,sera from liver failure rat and both mixture.To confirm the best condition for rMSCs to differentiate into hepatocytoid cell.[METHOD]We collected the bone marrow under sterile condition and cultured by blood-welt.To get rid of fibroblast and blood cells by density gradiend(ficoll1.077g/ml) centrifugation .After controlling digestion time we get more purify MSCs.They amplificated in L- DMEM with 10% FBS, 100 U/ mL penicillin and 100 U/mL streptomycin,MSCs were identified by immunocytochemistry and differentiation potency detection.Passages3 were induce into hepatocytoid cells with liver homogenate, sera from liver failure rat and both mixture .Then hepatocytoid cells were observed,and its expression of ALB was detected by immunocytochemistry ,glycogen by PAS(Periodic acid-Shiff reaction)AFP,ALB were detected by reverse transcription polymerase chain reaction(RT-PCR).[RESULT]1,The primary cultured was sand-like.After changing the medium,the welt cells were living behaviores and were quite stable in L-DMEM with 10% FBS .The cells morphology was the typical fibroblast-like and whirlpool-like or grass bunch when the density of cells increased.Sometimes the cells was triangle-like when the density decreased. After using density gradiend(ficoll1.077g/ml) centrifugation,the purity of MSC was evidently enhanced.2,Passage 3 MSCs,immunocytochemistry result showed CD105positive.3,Passage 3 differentiated to adioblast in adipogenic medium.Red O staining result was positive.4,Passage 3 differentiated into hepatocyte-like cells in three kinds of induction system.It has proved that induction system plays an important role in the differentiation of MSCs.Mixture is the best condition to induce the MSCs into hepatocyte.[CONCLUSION]1,It was a convenient method by blood–welt cuture and density gradient centrifugation for isolation and purification of MSCs from bone marrow in vitro .We gained MSCs with more purity and activity from observing cells morphology .2,Passage 3 MSCs,immunocytochemical result showed CD105positive.3,MSCs can different into adipocyte under some conditions.So MSCs can differentiate into various kinds of cells.4,We can detected its expression of AFP ALP by immunocytochemistry and RT-PCR.So it has been proved that MSCs can differentiate into hepatocyte-like cells with liver homogenate in vitro.The Mixture is the best condition to induce the MSCs into hepatocyte.