Expression of vascular endothelial growth factor gene mediated by lipofectamin in canine bone marrow

Objective To investigate the vascular endothelial growth factor (VEGF) secretion potential of canine bone marrow mesenchymal stem cells (BMSCs) that were transfected with hVEGF165 gene,and to provide scientific basis for stem cell treatment in avasctlar bone necrosis. Methods Marrow stromal cells were obtained from the tibia of adult dogs.The cells were isolated and differential cultured in vitro to obtain BMSCs.An hVEGF165 gene transfection group and a control group were set up.In the gene transfection group after passage the recombinant pcDNA32hVEGF165 plasmid was transfected into the BMSCs with lipofectamin and the transfection efficiency was observed under fluorescent microscope.For both groups VEGF165 expression levels in these BMSCs were quantified by ELISA at 1,3,5,7,9,11and 13 days.MTT method was applied to detect cell proliferation and draw the growth curve. Results Canine BMSCs were successfully transfected with hVEGF165 via lipofectamin and secreted VEGF proteins.VEGF secretion in culture media of hVEGF165 transfection group was higher than that of the control group ( P ＜ 0.05).ELISA detected increased VEGF secretion in 48 hours of canine BMSCs culture which peaked in 1week in the hVEGF165 transfection group.VEGF concentration was around 300 ng/L,being 10 folds of that of the control group.The difference was significant ( P ＜ 0.05).BMSCs growth curve of the transfection group and control group was not significantly different ( P ＞ 0.05 ).Conclusion hVEGF165 can be transferred into canine BMSCs using gene transfection technique.The transfected BMSCs express VEGF protein of bidogical activity.Cell proliferation is not affected. Key words: Vascular endothelial growth factor; Bone mesenchymal stem cells; Lipofectamin; Plasmids; VEGF165