Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells （MSCs） with human vascular endothelial growth factor 165 （hVEGFI65） gene and human...Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells （MSCs） with human vascular endothelial growth factor 165 （hVEGFI65） gene and human bone morphogenetic protein 2 （hBMP2） gene. Methods The hVEGF165 and hBMP2 cDNAs were obtained from human osteosarcoma cell line MG63 and cloned into lentiviral expression vectors designed to co-express the copepod green fluorescent protein （copGFP）. The expression lentivector and packaging Plasmid Mix were co-transferred to 293TN cells, which produced the lentivirus carrying hVEGF165 （Lv-VEGF） or hBMP2 （ Lv-BMP） , respectively. MSCs of Wistar rats were co-transfected with Lv-BMP and Lv-VEGF （BMP ＋ VEGF group）, or each alone （BMP group and VEGF group）, or with no virus （ Control group）. The mRNA and protein expressions of hVEGF165 and hBMP2 genes in each group were detected by real-time PCR and enzyme linked immunosorbent assay （ELISA）. Results Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed and confirmed by restriction endonucleses analysis and DNA sequencing analysis. A transfer efficiency up to 90% was archieved in all the transfected groups detected by the fraction of fluorescent cells using fluorescent microscopy. From the results generated by real-time PCR and ELISA, VEGF165 and BMP2 genes were co-expressed in BMP ＋ VEGF group. No significant difference of BMP2 expression was detected between BMP ＋ VEGF and BMP groups （ P 〉 0. 05）. Similarly, there was no significant difference of VEGF165 expression between BMP ＋ VEGF and VEGF groups （ P 〉 0. 05）. Conclusion VEGF165 and BMP2 genes were successfully co-expressed in MSCs by lentivirus-mediated co-transfection, which provided a further foundation for the combined gene therapy of bone regeneration.展开更多
基金Supported by Key Program of Shanghai Science and Technology Committee (054119520)
文摘Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells （MSCs） with human vascular endothelial growth factor 165 （hVEGFI65） gene and human bone morphogenetic protein 2 （hBMP2） gene. Methods The hVEGF165 and hBMP2 cDNAs were obtained from human osteosarcoma cell line MG63 and cloned into lentiviral expression vectors designed to co-express the copepod green fluorescent protein （copGFP）. The expression lentivector and packaging Plasmid Mix were co-transferred to 293TN cells, which produced the lentivirus carrying hVEGF165 （Lv-VEGF） or hBMP2 （ Lv-BMP） , respectively. MSCs of Wistar rats were co-transfected with Lv-BMP and Lv-VEGF （BMP ＋ VEGF group）, or each alone （BMP group and VEGF group）, or with no virus （ Control group）. The mRNA and protein expressions of hVEGF165 and hBMP2 genes in each group were detected by real-time PCR and enzyme linked immunosorbent assay （ELISA）. Results Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed and confirmed by restriction endonucleses analysis and DNA sequencing analysis. A transfer efficiency up to 90% was archieved in all the transfected groups detected by the fraction of fluorescent cells using fluorescent microscopy. From the results generated by real-time PCR and ELISA, VEGF165 and BMP2 genes were co-expressed in BMP ＋ VEGF group. No significant difference of BMP2 expression was detected between BMP ＋ VEGF and BMP groups （ P 〉 0. 05）. Similarly, there was no significant difference of VEGF165 expression between BMP ＋ VEGF and VEGF groups （ P 〉 0. 05）. Conclusion VEGF165 and BMP2 genes were successfully co-expressed in MSCs by lentivirus-mediated co-transfection, which provided a further foundation for the combined gene therapy of bone regeneration.
