Constuuction Of Recombinant Adenovirus Vectors Encoding Human BMP-2 Gene And Transfection On Bone Marrow Mesenchymal Stem Cells

Objectives:(1)To construct and identify the recombinant Adenovirus vectors encoding rat hBMP-2 gene.(2)To evaluate the effects of hBMP-2 gene transfection on bone marrow mesenchymal stem cells(BMSCs) in vitro.Methods:(1)The hBMP-2 gene sequence was amplified by PCR from human cDNA template and then cloned into shuttle plasmid pAV-MCMV-EGFP-3FLAG .The recombinant shuttle plasmid was constructed and transformed into E.coli DH5α.The positive colonies was identified by colonies PCR and sequencing. The expression vetors were amplified and packaged . The expression vetors and backbone plasmids were co-transfeced and amplified in 293 cells.The reccombination was identified by PCR and western blot. The infectious titer of the recombinant Adenovirus was tested by dilution and end point method.(2)BMSCs were harvested from bone marrow of SD rat. CD90 and CD45 were tested by flow cytometry.The successfully packaged virus were transfected into BMSCs and expression of hBMP-2 gene was confirmed by EGFP expression under the fluorescence microscope.Elisa were carried out to monitor the expression of hBMP-2 and ALP protein in mesenchymal stem cells and finally evaluated the effect of hBMP-2 on the proliferation of BMSCs by MTT assay.Resules:(1)The resulting 1223 bp hBMP-2 gene fragment was confirmed by colonies PCR and sequencing.(2)After amplified in 293 cells,the virus titre is 5×1010 pfu/ml .(3)BMSCs were successfully harvested from bone marrow of SD rat and identified by flow cytometry.After primary culture of 7-10 days,cultured cells displayed typical fusiform shape and the growth status was like"cobblestones"or"whirlpool"under light microscope and could be differentiated into osteoblast cells,adipocyte and neuron-like cells in vitro. (4)After the transfection of hBMP-2 gene, the expression of hBMP-2 and ALP protein by BMSCs was detected by Elisa.MTT assay suggested that hBMP-2 protein expression in the BMSCs has a significant effect on the proliferation of BMSCs(p<0.05).Conclusion:The recombinant Adenovirus carrying hBMP-2 gene had been constructed successfully and was transfected into BMSCs stably at high efficiency. Moreover, the expressions of hBMP-2 and ALP have a significant effect on BMSCs in vitro.