Zhi-bin Shi1, Kun-zheng Wang. 1. Department of Orthopedics, The Second Affiliated Hospital of Xi'an Jiaotong University, Xiwu Road, Xi'an 710004, Shaanxi Province, China. jackky9999@sohu.com
Abstract
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 MTT 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, VEGF165, and BMP(7) 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 BMP(7) proteins expressed from the vector have efficient biological activity in vitro.
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 MTT 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, VEGF165, and BMP(7) 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 BMP(7) proteins expressed from the vector have efficient biological activity in vitro.
Authors: María Piedad Ramírez-Fernández; José Luis Calvo-Guirado; José Eduardo-Maté Sánchez de-Val; Rafael Arcesio Delgado-Ruiz; Bruno Negri; Guillermo Pardo-Zamora; David Peñarrocha; Cristina Barona; Jose Manuel Granero; Miguel Alcaraz-Baños Journal: Clin Oral Investig Date: 2012-02-11 Impact factor: 3.573