Influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2-vascular endothelial growth factor 165 in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells

Abstract

Objective To observe the influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2 (BMP2)-vascular endothelial growth factor 165 (VEGF165) in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods The isolated rabbit BMSCs were used as the research objects, and the recombinant plasmid pIRES-BMP2-VEGF165 was transfected into cells in the presence of liposomes. Then, G418 was used to screen stable cell lines in vitro and mRNA and protein levels were detected for transfection efficiency. Stable cell lines were then cultured and the cells were divided into pIRES group, pIRES-BMP2-VEGF165 transfection group and osteogenic induction group. Western blotting was used to detect the expression of Collagen Ⅰ (ColⅠ) and osteocalcin (OCN) protein at 7th and 21st day after cultivation, respectively. After 14 days of cultivation, gomori modified calcium and cobalt staining was used to detect alkaline phosphatase activity. After 21 days of cultivation, calcified nodules of cells were detected by alizarin red method calcium staining. Results Compared with the empty plasmid pIRES group, the expression of BMP2 and VEGF165 was detected at the mRNA and protein levels of pIRES-BMP2-VEGF165 plasmid group. Compared with empty plasmid group and osteogenic induction group, the expression levels of ColⅠ and OCN protein in recombinant plasmid group were significantly increased (Col Ⅰ: 0.597±0.040 vs. 0.230±0.020, P=0.000; 0.597±0.040 vs. 0.457±0.035, P=0.005; OCN: 0.433±0.038 vs. 0.083±0.021, P=0.000; 0.433±0.038 vs. 0.350±0.036, P=0.046). Positive relative area (%) of alkaline phosphatase staining increased significantly (69.000±5.568 vs. 18.333±3.056, P=0.000; 69.000±5.568 vs. 43.333±5.508, P=0.002). Positive relative area (%) of calcified nodules increased markedly (37.747±3.763 vs. 1.587±0.972, P=0.000; 37.747±3.763 vs. 19.330±4.033, P=0.008). Conclusion Human recombinant eukaryotic expression plasmid pIRES-BMP2-VEGF165 was successfully transfected into BMSCs. As compared with osteogenic induction group, the recombinant plasmid pIRES-BMP2-VEGF165 was more effective in osteogenic differentiation of the cells. Key words: Bone mesenchymal stem cells; pIRES-bone morphogenetic proteins 2 -vascular endothelial growth factor 165 plasmid; Transfection in vitro; Bone morphogenetic proteins 2; Vascular endothelial growth factor 165