Effects of recombinant adeno-associated viral vectors on angiopoiesis and osteogenesis in cultured rabbit bone marrow stem cells via co-expressing hVEGF and hBMP genes: A preliminary study in vitro

摘要

Objective: VEGF and BMP play important roles in angiogenesis and osteogenesis. Combining these two factors may be a promising therapeutic strategy for avascular necrosis of the femoral head (ANFH).Methods: Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and purified by density gradient centrifugation combined with attachment culture methods. The purity and characteristics of the BMSCs were detected by cell surface antigen identification. The best MOI of BMSCs transfected with rAAV was detected by fluorescent cell counting, and cell viability was determined by MU assay. Expression of the genes of interest was detected by GFP gene expression, RT-PCR assay, and ELISA assay. The biological activities of VEGF and BMP were detected by angiogenic and osteogenic assays.Results: The best MOI of BMSCs transfected with rAAV was 5 x 10(4) v.g./cell. Cell growth curves showed vigorous cell viability. Expressions of the GFP, VEGF(165), and BMP, genes were detected 1 day post-transfection and peaked 14 days post-transfection. Expression of the genes of interest was sustained over 1 month. VEGF and BMP proteins secreted from BMSCs transfected with rAAV-hVEGF(165)-IRES-hBMP(7) enhanced angiogenesis and osteogenesis in vitro.Conclusion: Recombinant adeno-associated viral vectors co-expressing the hVEGF(165) and hBMP(7) genes showed efficient gene expression ability. The VEGF(165) and BMP7 proteins expressed from the vector have efficient biological activity in vitro