Biological Characteristics Of Rat Bone Marrow Mesenchymal Stem Cells And The Repair Of Acute Chemical Injured Liver

一,Isolation,Cultivation,Identification and EGFP Marking of Rat Bone Marrow Mesenchymal Stem CellsObjectives:This research was to intent of isolation and purification in vitro of BMSCs from rat bone marrow.Some biological characteristics of BMSCs were identified for the base of cells therapy;To investigate the transduction and gene expression of different lentivirus vectors in the rat BMSCs in vitro,in order to establish fundament for observe cell differentiation to hepatocyte in vivo.Methods:Myeloid cells of Hindlimb bone were collected in Wistar male rat at the age of 4 weeks,weighting 100～120g in asepsis condition.BMSCs were isolated by gradient centrifuging method and adherent culture in vitro.The adherent cells were cultured in DMEM containing 10%～15%FBS after 24 hours and incubated at 37℃in 5%CO₂.once every three days to replace the culture medium.As 80%～90 %of cells are fused,the cells were dissociated with 0.25%trypsin and passage,so that the cells gradually purification.1.Observation and identification of BMSCs: (1)The form and size were observed under phase-contrast microscope;(2) ultra-structure were observed under transmission electron microscope;(3)MTT assay was used to measure cell growth curve;(4)the expression of CD29,CD34,CD45,CD90 were detection by flow cytometry.2.Then add harvested lentiviral supernate to experiment groups by multiplicity of infection(MOI)(1,10,30,50,70,100), use PBS as control.Observe the expression of EGFP and morphologic change of labeled BMSCs by fluorescence microscope and account the transfection efficiency to find the best MOI.Results:1.According to the method as described above for the isolation of mononuclear cells from bone marrow using 1.073g/mL percoll density centrifugation and cultured adherence,the morphology of BMSCs was spindle-like bearing round or oval-shape nuclei;the result of flow cytometry:CD29+/CD90+/CD34-/CD45-;2.Growth curve of passage cells showed that there were difference of latency,of activity and flat periods.3.Transmission electron microscopy showed that there were two of BMSCs,the small of both were infantile cells.4.The expression of EGFP on BMSCs began at 24^th hour after transduction, reaching the maximum at the 5^th to 7^th day,the highest efficiency of which is (79.85±4.92)%(MOI=30,day7).The efficiency of transduction increased as the increasing of MOI.The efficiency of transduction when MOI equaled to 30,50,70,100 was obviously higher than the effect of that when MOI equaled 1,10(P＜0.05).Cell morphology has no significant different from controls.The best MOI was found to be at least MOI=30,and EGFP expression continued more than 14 days.Conclusions:BMSCs were gained through isolation,purification and identification of BMSCs in vitro in this study,and some biological characteristics of BMSCs were identifical..Lentiviral vector system with EGFP gene can successfully transfect BMSCs and acquired highly and long time stable EGFP expression and this transduction does not bring any effect on cell biological characteristics,in order to establish fundament for observe BMSCs differentiation to hepatocyte in vivo.二,Study on the Repair of Allogeneic Graft Bone Marrow Stem Cells with EGFP on Acute Chemical Injured LiverObjectives:Provide a report on the feasibility to track the GFP-labeled BMSCs in the rat liver and ultimately build a theoretical basis for treating the acute injured livers on the basis of allogeneic grafted BMSCs.Methods:Thirty Wistar rats,6-weeks old,weighting 150g～180g,they were randomly divided into six groups after raised one week in separated cages,the model of acute injuried groups was constructed by 2%carbon tetrachloride.A group(normal control group):normal rats,no cell transplantation,PBS was injected by tail vein;B group(normal + no marked group ):normal rats,transplanted to unlabeled BMSCs by tail vein;C group(normal + marked group):normal rats,transplanted to GFP-labeled BMSCs by tail vein;D group(liver injury control group):liver damaged rats,no cell transplantation,PBS was injected by tail vein;E group(liver injury + no marked group):liver injuried rats,transplanted to unlabeled BMSCs by tail vein;F group(liver injury + marked group):liver injuried rats,transplanted to GFP-labeled BMSCs by tail vein.The rats were sacrificed at 1wk after transplantation,liver function was examined,livers were prepared for hematoxylin and eosin stain, examined by optic and fluorescent microscope.Results:1.The levels of ALT,AST,TBIL in the serum were significantly different between the rats(D,E,F) that fed with CCL₄ for 1 week and those normally fed(A,B,C).The levels of ALT,AST,TBIL were significantly higher than those of the normally fed rats(P＜0.05).Cellular necrosis,bridging necrosis,congestion in the hepatic sinusoid, and infiltration of inflammatory cells were seen in the injured livers.2.After BMSCs grafting for one week,the levels of ALT,AST,TBIL in the serum were ameliorated significantly,compared with those without BMSCs grafting(P＜0.05).Meanwhile,the cellular necrosis,congestion,infiltration of inflammatory cells were also ameliorated compared with the livers without BMSCs grafting.However,there were no differences between the EGFP-labeled BMSCs and non-EGFP-labeled BMSCs group in view serological examination,histopathological examination.F group could find EGFP+ cells in fluorescence microscope.Some of these cells have migrated into parenchyma.Conslusions:1.Verified feasible method of rat liver injuried model-build.2.The damaged liver function in the model of chemically induced acute liver injury can be repaired by BMSCs implantation.3.EGFP neither affects the liver function of normal rats,nor hinders the role of BMSCs in repair of the injured liver function in the model of chemically induced acute liver injury.