hVEGF_(165)及hBMP-7共表达重组腺相关病毒载体介导基因表达的时效性研究

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

目的探讨hVEGF165及hBMP-7共表达重组腺相关病毒载体(recombinant adeno-associated virus,rAAV)介导基因表达的时效性,为体内应用其进行骨坏死的基因治疗奠定理论基础。方法应用荧光细胞计数法测定rAAV转染兔BMSCs的最佳转染复数。分别采用rAAV-hVEGF165-内部核糖体进入位点序列(internal ribosome entry site,IRES)-hBMP-7(实验组)、绿色荧光蛋白(green?uorescent protein,GFP)标记平行对照rAAV-IRES-GFP(对照组)在最佳转染复数条件下转染体外培养的第3代兔BMSCs。于不同时间点分别行GFP表达检测、RT-PCR检测、Western blot检测明确rAAV体外介导基因表达的时效性关系。将实验组及对照组病毒转染后的BMSCs以5×106个/mL密度,分别注射至9只2月龄纯种雄性新西兰大耳白兔制备的肌袋模型内各1mL(实验组1及对照组1),于不同时间点分别行GFP表达检测、Western blot检测及ELISA检测明确rAAV体内介导基因表达的时效性关系。结果 rAAV转染兔BMSCs的最佳转染复数为5×104v.g/cell。通过体外检测显示rAAV转染BMSCs后1d即有目的基因表达,14d时表达强度明显增强,至28d时仍维持较强表达。通过体内检测显示体内转染2周可检测到目的基因表达,6周时表达强度增强,8周时仍维持较高水平。ELISA法检测实验组1细胞培养上清中hVEGF165和hBMP-7含量分别为(248.67±75.58)pg/mL和(4.80±0.61)ng/mL,对照组1分别为(32.28±8.42)pg/mL和(0.64±0.42)ng/mL,两组比较差异均有统计学意义(P<0.05)。结论双基因共表达rAAV-hVEGF165-IRES-hBMP-7具有较好的转染时效性。

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 × 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 × 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.