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.
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.


CONCLUSIONS
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.
Shi
Zhi-bin
ZB
Department of Orthopedics, The Second Affiliated Hospital of Xi'an Jiaotong University, Xiwu Road, Xi'an 710004, Shaanxi Province, China. jackky9999@sohu.com
Wang
Kun-zheng
KZ
D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism*; D055419:Bone Morphogenetic Protein 7 / Q000096:biosynthesis*; D002470:Cell Survival; D000229:Dependovirus / Q000235:genetics; D005822:Genetic Vectors; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000235:genetics*; D010012:Osteogenesis / Q000235:genetics*; D011817:Rabbits; D014161:Transduction, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis*
D055419:Bone Morphogenetic Protein 7; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A