| Objective:The chimeric liver model was established to provide an experimental animal model for the study of the evolution of mesenchymal stem cells(MSCs)in vivo,the mechanism of immune tolerance and the development of human liver diseases.Method:1.Isolation and identification of cells of goat bone marrow mesenchymal stem:young male goat bone marrow under aseptic conditions,0.5-1 ml1 months old,by whole bone marrow adherent culture method were cultured with morphological observation,immunohistochemical analysis,CD13,CD29,CD44,CD90,CD105 and CD34 antibody labeled bone marrow mesenchymal stem cells,identified by flow cytometry;in vitro differentiation and identification of the.2.Goat BMMSC markers:a lentiviral vector carrying a green fluorescent protein gene(GFP)was co cultured with BMMSC to label BMMSC,and the optimal dose of lentivirus was determined.3.The GFP labeled BMMSC was injected into the liver of fetuses.The technique of injecting GFP labeled BMMSC into the liver of fetal sheep was established by B-mode ultrasound to observe the life status of the recipient ewes after transplantation.4.Goat chimeric liver model identification:50 days after delivery of the pregnant sheep,the part of liver tissue of the new lamb was cut by surgical method,and the following identification analysis was carried out:(1)GFP positive cells were observed:.the liver was harvested from the lamb and frozen sections were made.The presence and distribution of GFP positive cells in liver tissue were observed under fluorescence microscope;(2)HE staining analysis:the frozen sections were fixed in formalin,and stained with 5min.After HE staining,the distribution of GFP positive cells and the pathological structure were observed under the microscope;(3)Identification of SRY gene expression:a small amount of liver tissue of newborn lamb was collected,respectively.DNA was extracted and amplified by quantitative PCR to identify the expression of SRY gene.(4)The proportion of GFP positive cells were cut out a little lamb liver cut,with fresh collagenase digestion,liver into single cell suspension.Flow cytometry was used to analysis the proportion of GFP positive cells.Results1.By whole bone marrow adherent culture method,using DMEM/F12 suspension rinse containing 10%fetal bovine serum after bone marrow cells and static culture for 4-5 days,to be adherent long spindle cells or cells were observed under microscope at the colony,can be observed under the microscope were densely arranged fibroblast like cells a spiral of adherent 8-12 days.The third generation cells were detected by flow cytometry.The results showed that these' cells expressed high levels of CD13,CD29,CD44,CD90,CD 105,low expression or no expression of CD3.It can be induced to differentiate into adipocytes,osteocytes and chondrocytes,and the cells obtained are judged to be BMMSC.2.By 0,10 and 100 MOI with lentivirus and goat BMMSC were cultured for 3 days to green fluorescence under a fluorescence microscope,that has been successfully transfected into GFP,and the MOI transfection group the expression of GFP 100 was significantly higher,namely green fluorescent brightness strong,MOI transfection group the expression of GFP 10 less,MOI for the control group of 0 no green fluorescence was observed.The GFP transfected BMMSC were subcultured by detecting the expression rate of GFP cells after passage,flow cytometry,experimental results showed that 1-4 the transfection rate was(42.3 +2.25)%and(41.6 + 2.65)%and(41.4 + 3.75)%and(38.2 + 4.75)%,the expression of GFP in four generations there was no significant difference.3.In ultrasonic imaging conditions,can be observed in different gestational age rhythm of pregnant ewe belly of a fetal heart beat,can be accurately determined in the fetal heart next to the fetal liver,and a one-time injection of 0.5ml mesenchymal stem cells to 2-4 months fetal liver,does not lead to excessive abortion,obtained 5 newborn lamb.4.Identification of chimeric liver models in goats:(1)The frozen sections observed under fluorescence microscopy injected cells in liver tissue with green fluorescence distribution,some tubular structure rules,and more standard injection of mesenchymal stem cells in liver tissue,lamb had more GFP positive tubular structure.(2)In frozen section,GFP positive cells can be observed in the same field of view,the central vein and the surrounding liver around the liver,liver sinusoids and other normal liver histological structure,cell morphology is normal.GFP positive cells have been embedded in liver tissues and transformed into liver tissue cells.Similarly,GFP positive tubular structures were observed in the frozen sections.By HE staining indicated that is typical of the bile duct,there are many columnar epithelial cells surrounded by dense tubular structure,the nucleus is deep blue,bile duct surrounding the typical structure of the portal area can be observed,and no obvious lymphocyte infiltration.These results indicated that GFP markers of fetal liver in the injection of BMMSC in lamb within 50 days after birth were not immune cells in liver tissue,fitted to the lamb evolved into liver cells or bile duct columnar epithelial cells and the formation of bile duct structure.(3)The expression of SRY gene identification:PCR method was used to detect the expression of SRY gene in liver tissue of newborn lamb injected mesenchymal stem cells 50 days after birth,including 2 female newborn lamb,and inject more mesenchymal stem gene expression detected more SRY cells in the liver tissue of female lamb.(4)The proportion of GFP positive cells in the liver tissue of newborn lamb:Determination of injected cells 50 days after birth,by flow cytometry to detect the presence of GFP positive cells,at least 2.25%of the GFP cells,with a maximum of 7.23%GFP cells.Conclusion1.The bone marrow 0.5-lml of 1 month old lambs was cultured by the whole bone marrow adherent culture method and cultured by 10%FBS-DMEM/F12.The MSCs could be isolated and expanded into a vortex like and densely arranged mesenchymal stem cell;2.By carrying green fluorescent protein(GFP)lentivirus and mesenchymal stem cells co culture method can be successfully labeled mesenchymal stem cells,the best MOI lentivirus was 100,labeled cells after 4 generations the expression rate of GFP was not significantly reduced;3.Under ultrasound guidance,can achieve the March old fetal goat liver parenchyma mesenchymal stem cells injection,and the one-time injection of 0.5ml mesenchymal stem cells(including 106-108)to be 2-4 months fetal liver in ewes abortion;4.The chimeric liver can be formed by injecting GFP labeled allogeneic goat mesenchymal stem cells into the fetal liver at different ages.The presence or evolution of GFP positive cells into columnar epithelial cells and the formation of bile duct structures were observed in the liver pathological tissue 50 days after the birth of the lambs.Can be identified by PCR.The expression of SRY mRNA was detected in newborn female lamb liver tissue,analysis showed that the injection of multicellular GFP positive cells in the liver of flow cytometry showed that successfully established liver allograft goat chimera model. |
PDF Full Text Request