Abstract:

Objective To investigate the effects of lentivirus-mediated co-transfection of bone morphogenetic protein 2 (BMP2) gene and vascular endothelial growth factor 165 (VEGF165) gene on osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (MSCs). Methods The expression lentivirus vectors carrying VEGF165，BMP2 or green fluorescent protein (GFP) gene were constructed respectively, and recombinant lentivirus carrying VEGF165 (Lv-VEGF), BMP2 (Lv-BMP) or GFP (Lv-GFP) were packaged and produced respectively. Rat bone marrow-derived MSCs were isolated and cultured in vitro, and were transfected with Lv-VEGF (VEGF group), Lv-BMP (BMP group) or Lv-GFP (GFP group), co-transfected with Lv-VEGF and Lv-BMP (BMP+VEGF group), or transfected with no virus (control group). The expression of VEGF165 and BMP2 mRNA 7 d after transfection and that of osteocalcin (OCN) mRNA 1, 2 and 4 weeks after transfection in MSCs was detected by RT-PCR in each group. The expression of VEGF165 and BMP2 protein 1, 4 and 8 weeks after transfection and that of OCN protein 1, 2 and 4 weeks after transfection in supernatant fluid of culture was detected by ELISA in each group. Alkaline phosphatase (ALP) staining was conducted, and ALP activity of MSCs was measured 14 d after transfection in each group. Results VEGF165 and BMP2 mRNA and protein effectively coexpressed in BMP+VEGF group. There was no significant difference in the expression of BMP2 mRNA and protein between BMP+VEGF group and BMP group (P>0.05), and there was also no significant difference in the expression of VEGF165 mRNA and protein between BMP+VEGF group and VEGF group (P>0.05). There was no expression of BMP2 mRNA and protein in control group, GFP group and VEGF group, and there was also no expression of VEGF165 mRNA and protein in control group, GFP group and BMP group. The expression of OCN mRNA and protein in BMP+VEGF group was significantly higher than that in the other groups (P<0.01). The area with positive staining of ALP in BMP+VEGF group was larger than that in control group, VEGF group and BMP group, and the ALP activity in BMP+VEGF group was significantly higher than that in the other groups (P<0.01). Conclusion Lentivirus-mediated co-transfection of BMP2 gene and VEGF165 gene may promote osteogenic differentiation of bone marrow-derived MSCs.

Key words: bone morphogenetic protein 2, vascular endothelial growth factor 165, co-transfection, lentiviral vector, osteogenic differentiation