| The isolation, cultivation, identification and induction of mesenchymal stem cells of Japan big-eared white rabbit.Objective:Isolation,cultivation of the mesenchymal stem cells in Japan big-eared white rabbit in vitro, the well proliferated cells were selected in this trial.Methods:Adopting health juvenile Japan big-eared white rabbit, anesthesia lethal after flushing bone marrow collected bone marrow liquid.Bone marrow mononuclear ceps were isolated with density gradient centrifugation and adherence screening method.BMSCs were obtained by removing the non-adherent cells and observed with light microscope. Then the BMSCs were purified and expanded through passaging in time. The four surface antigen:CD34,CD44,CD45, CD90 of cells in the third passage were tested with flow cytometry. ALP dye and oil red O staining were used to identify the potentials in differentiating into osteoblasts and adipocytes.Results:The density of the third and following passages of rabbit MSCs was idendical. Cells appeared in spindle shape, as the fibroblast cells. The third generation of MSCs by flow cytometry technique identified cells CD34 (-), CD45 (-), CD44 (+),CD90 (+) with MSCs surface antigen.After appropriate induction, ALP and oil red d staining were both positive in the cells.The shape of the cells by long shuttle deformation for polygon.Conclusion:With the methods of density gradient centrifugation and adherence screening, we can obtain high purified rabbit MSCs. The isolated BMSCs express the mesenchymal stem cells specific surface antigens. BMSCs can induce differentiation of osteoblasts or adipocytes.Construction of TGF-β1 Over-expression Lentivirus VectorsObjective:Packaging TGF-β1 gene to lentiviral vector system.Methods:Using gene cloning technology to directional TGF-β1 gene cloning to slow virus vector, build the TGF-beta 1 gene recombinant plasmid, and to identify the PCR, enzyme digestion and sequencing. Under the action of LP2000 liposome transfection 293 T cells, from cell culture is rich in slow virus particles, for its high concentration after the drop degree of lentivirus concentrate, to real-time fluorescence QPCR detection slow drop degree of virus.Results:Gene cloning technology was adopted to construct the TGF-beta 1 recombinant lentiviral vector,which was identified by the polymerase chain reaction (PCR),enzyme digestion and sequencing. The transfected 293T cells were identified to express correctly.Conclusion:Gene cloning technology can successfully build TGF-β1 restructuring slow virus vector, transfection 293t cells can express correctly.TGF-β1 gene transfection MSCs and observe its expression in the MSCsObjective:Use TGF-β1 genetic modification MSCs, observe the TGF-β1 gene expression in the MSCs effects on the proliferation and differentiation.Methods:Through preliminary experiment to determine the appropriate MOI, slow virus infection and with appropriate MOI infected rabbit MSCs.Fluorescence microscope TGF-beta 1 protein expression and infection, and semi-quantitative rt-pcr and Wstern Blot detecting infected rabbit TGF-beta 1 mRNA and Collagen in the MSCs Ⅱ expression.Observation of BMSCs after transfection cell proliferation and metabolism ability.Results:The optimum MOI of BMSCs was 108 with the 1×108TU/ml lentiviral titer. rt-pcr and Wstern Blot from the infected rabbit MSCs can be detected TGF-beta 1 mRNA and Collagen Ⅱ expression.Conclusion:the gene cloning techniques to build TGF-beta 1-pCDH-GFP fusion gene recombinant lentivirus vectors transfection after MSCs, TGF-beta 1 and Collagen Ⅱ in the MSCs can efficiently express, and MSCs, proliferation and metabolic activity... |
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