Expression of recombinant adenovirus-mediated VEGF_(165) in bone marrow stromal cells of rats

Objective To construct recombinant adenovirus vector carrying vascular endothelial growth factor 165(Ad-VEGF165),amplify the adenovirus vector in 293T cells,transfect Ad-VEGF165 into the bone marrow stromal cells(bMSCs) of Wistar rats,and then to assay the expression of VEGF165.Methods VEGF165 obtained by PCR was digested and inserted into adenovirus shuttle plasmid pAdTrack-CMV to generate recombinant plasmid pAdTrack-VEGF165,and then the Pme I-linearized plasmid pAdTrack-VEGF165 was electroporated into E.coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1.The identified recombinant plasmid pAdEasy-VEGF165 DNA was digested with Pac I and transfected into 293T cells to package adenovirus,followed by identification of the recombinant adenovirus by means of observation of the enhanced green fluorescence protein(EGFP) expression under fluorescent microscope.After amplified in 293T cells,the obtained adenovirus were transfected into 293T cells again,and EGFP expression was detected.Ad-VEGF165 transfected bMSCs were cultured in vitro.The expression of VEGF165 in bMSCs after transfection was determined by observing the expression of EGFP and detected by RT-PCR.ELISA method was applied to assay the secretion of VEGF165.Results Recombinant adenoviral VEGF165 was constructed successfully,which was confirmed by restriction enzyme digestion,gene sequencing and EGFP expression.EGFP expression could be observed under fluorescent microscope,and the expression of VEGF165 was confirmed by RT-PCR.ELISA analysis showed the quantity of expression of VEGF165 in transfection group was higher than that in control group(P0.01).Conclusion Ad-VEGF165 has been successfully constructed,which may provide a basis for VEGF165 gene therapy.Exogenous VEGF165 can be expressed in bMSCs,which may offer the possibility of a novel approach to local gene therapy of bone tissue engineering.