[Study on time effect of gene expression of recombinant adeno-associated virus vector co-expressing human vascular endothelial growth factor 165 and human bone morphogenetic protein 7 genes].

OBJECTIVE: To study the time effect of the gene expression of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothelial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes so as to lay a theoretical foundation for gene therapy of osteonecrosis. METHODS: The best multiplicity of infection (MOI) of BMSCs transfected with rAAV was detected by fluorescent cell counting. The 3rd generation rabbit bone 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), respectively. The expression of GFP was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by RT-PCR assay and Western blot assay in vitro. The transfected cells in 2 groups were prepared into suspension with 5 x 10(6) cells/mL, and injected into the rabbit thigh muscles of experimental group 1 (n=9) and control group 1 (n=9), respectively. The muscle injected with rAAV-IRES-GFP was sliced by frozen section method and the expression of GFP protein was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by Western blot assay and ELISA assay in vivo. RESULTS: The best MOI of BMSCs transfected with rAAV was 5 x 10(4) v.g/cell. In vitro, the expressions of GFP, hVEGF165, and hBMP-7 genes started at 1 day after transfection, the expressions obviously increased at 14 days after transfection, and the expression maintained the strong level at 28 days after transfection. In vivo, the expressions of GFP, hVEGF165, and hBMP-7 genes could be detected at 2 weeks after injection, and strong expressions were shown at 6 to 8 weeks after injection. The values of hVEGF165 and hBMP-7 were (248.67 +/- 75.58) pg/mL and (4.80 +/- 0.61) ng/mL respectively in experimental group 1, and were (32.28 +/- 8.42) pg/mL and (0.64 +/- 0.42) ng/mL respectively in control group 1; showing significant differences between 2 groups (P < 0.05). CONCLUSION: The rAAV-hVEGF165-IRES-hBMP-7 has efficient gene expression ability.