Study on transfection of dog bone mesenchymal stem cells with BMP-2/VEGF165-based lentivirus vector

Abstract

Objective To transfect the dog bone mesenchymal stem cells (BMSCs) with lentivirus-mediated vascular endothelial growth factor 165 (VEGF165) and bone morphogenetic protein 2 (BMP-2) and observe the expressions of VEGF165 and BMP-2 in BMSCs. Methods Lentivirus vectors carrying VEGF165 and BMP-2 genes were constructed and used to transfect dog third-generation BMSCs, including untransfected group, VEGF165 group, BMP-2 group and double-gene (VEGF165+ BMP-2) group. Expressions of VEGF165 and BMP-2 mRNA were detected by RT-PCR 7 d after the transfection.Expressions of VEGF165 and BMP-2 protein were detected by Western blotting 5 d and 28 d after the transfection. Cell growth was detected by MTT assay. Results RT-PCR showed high expression of VEGF165 mRNA in VEGF165 and double-gene groups, and BMP-2 mRNA in BMP-2 and double-gene groups, but there was no significant difference in group comparison(P>0.05). Western blotting showed specific performance of VEGF165 protein in VEGF165 and double-gene groups, and BMP-2 protein in BMP-2 and double-gene groups. In MTT assay cells were found to have 2-3 d relatively stationary phase in the early period, proliferated rapidly at 3 d, reaching the logarithmic growth, and entered the plateau phase with slow propagation. There was no significant difference in absorbance value before and after the transfection (P>0.05). Conclusion BMSCs with highly expressed VEGF165 and BMP-2 can be successfully obtained by lentiviral transduction. Key words: Tissue engineering; Mesenchymal stem cells; Lentivirus; Transfection