Objective: To study the biological activity of recombinant adeno-associated virus vector (rAAV) co-expressing human vascular endothelial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes in vitro so as to provide a new method for the therapeutics of osteonecrosis.
Methods: The 3rd passage rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group). The expressions of hVEGF165 and hBMP-7 were detected by ELISA assay at the 1st, 2nd, 3rd, 7th, 14th days and Western blot assay at the 14th day after transfection. The expression consistencies of hVEGF165 and hBMP-7 were observed by immunofluorescence assay at the 14th day after transfection. The biological activity of hVEGF165 was assessed by angiopoiesis experiment of the 3rd passage human umbilical vein endothelial cells (HUVEC). The biological activity of hBMP-7 was assessed by mineralization of BMSCs detected by ALP staining and alizarin red staining.
Results: With infecting time, the hVEGF165 and hBMP-7 expressions increased gradually in two groups, showing significant difference between two groups (P < 0.05). The expressions of hVEGF165 and hBMP-7 were positive in experimental group and negative in control group, respectively. Immunofluorescence assay showed positive expressions of hVEGF165 and hBMP-7 in the experimental group and negative expression in the control group, the expression of hVEGF165 and hBMP-7 had good consistencies. hVEGF165 secreted from BMSCs enhanced HUVEC migration, proliferation and tube formation in experimental group. There was significant difference in the number of blood vessel between two groups (P < 0.05). The ALP staining showed more strongly stained granules in experimental group than in control group. There was significant difference in the number of the mineralized nodules between two groups (P < 0.05).
Conclusion: The rAAV-hVEGF165-IRES-hBMP-7 has good biological activity in vitro.