The Research Of Bone Marrow Mesenchymal Stem Cells Modified By Transforming Growth Factor Beta 1

Isolation, cultivation, and identification of Beagle bone marrow mesenchymal stem cellsObjective:To establish a method for isolation, cultivation and identification of beagle bone marrow mesenchymal stem cells (BMSCs) in vitro, the well proliferated cells were selected to provide the basis for the transplantation experiment.Methods:10 ml bone marrow was extracted from the iliac of beagle by puncture in axenic condition. Combined with density gradient isolation and adherent culture method and enlarge the beagle BMSCs were isolated and cultured in vitro, and the induced BMSCs in the direction of osteoblast differentiation. Inverted phase contrast microscope to observe the morphological changes, draw the growth curve, flow cytometry instrument to detect cell surface antigens.Results:The primary and passaged BMSCs were spindle-shaped with adhesion characters, and grew in colonies. The growth curves of passage 1,3 and 5 were quite similar, exhibiting a large expansive potential. These cells were positive for essential BMSCs surface molecules including CD29 and CD44, and was negative for CD34. The BMSCs could be differentiated towards osteogenic phenotypes.Conclusion:BMSCs were isolated and cultured successfully from beagle ilium bone marrow by combination of density gradient isolation and adherent culture.Construction of TGF-β1 Over-expression Lentiviral Vector And Expression of TGF-β1 Gene in Beagle Bone Mesenchymal Stem CellsObjective:Packaging and transfecting TGF-β1 gene to BMSCs via lentiviral vector system, observe the TGF-β1 gene expression in the MSCs effects on the proliferation.Methods:The TGF-β1 gene was recombined to lentiviral vector by gene transfection technique. The combined plasmid was identified by polymerase chain reaction(PCR),enzyme digestion and sequencing and transfected to 293 T cells mediated by Iipofectamin2000 to produce lentiviral particles. The lentiviral titer was detected by real-time fluorescence quantitative PCR(RT Q-PCR) and was transducted to beagle BMSCs by optimum MOI. The expression of TGF-31 protein was observed with fluorescent microscope. We adopted semiquantitative RT-PCR and western blot to analyze the expression of TGF- β1 mRNA and protein. To observe the proliferation and metabolism of BMSCs cells after transfection.Results:Gene transfection technique was adopted to construct the TGF-β1 gene 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. The optimum MOI of BMSCs was 10 with the 1×108TU/ml lentiviral titer and BMSCs infected by TGF-β1. gene recombinant lentiviral vector can express TGF-β1 efficiently.Conclusion:Lentiviral vector mediated TGF-β1 gene can be successfully transfected into beagle BMSCs and can be highly efficient and stable expression.