重组腺病毒介导hCGPx转染致血管内皮细胞产生抗氧化损伤作用的研究

Recombinant adenovirus-mediated human cytosolic glutathione peroxidase gene transfection protects vascular endothelial cells from oxidative damage

目的研究重组腺病毒介导的人胞浆型谷胱甘肽过氧化物酶(hCGPx)转染对血管内皮细胞ECV304氧化损伤保护作用。方法将含hCGPxcDNA的质粒pGEM-T-hCGPx和重组腺病毒载体pACCMV-pLpA穿梭质粒进行基因重组,构建成pACCMV-hCGPx穿梭质粒后,与包装质粒pJM17共转染293细胞,构建成重组腺病毒AdCMV-hCGPx。用AdCMV-hCGPx转染体外培养的ECV304细胞并分为转染24、48和72h组,以转染空载体的细胞为对照组,检测转染细胞的基因表达水平。各组ECV304细胞经H2O2氧化损伤处理后,分别对细胞的活力和凋亡进行检测分析。结果各转染组细胞转染基因表达率均显著高于对照组(P<0.01)。经H2O2氧化损伤处理后,AdCMV-hCGPx转染组细胞活力较对照组明显增强,凋亡受到抑制。结论重组腺病毒介导的hCGPx转染可保护ECV304细胞抵抗氧化损伤,具有明确的细胞保护作用,其具体保护机制可能与抗氧化和抑制细胞凋亡有关。

Objective To study the protective effect of recombinant adenovirus-mediated human cytosolic glutathione peroxidase （hCGPx） gene transfection on vascular endothelial cells ECV304 from oxidative damage. Methods pGEM-T Easy Vector containing hCGPx cDNA and recombinant adenovirus shuttle plasmid pACCMV-pLpA were used to construct the shuttle plasmid pACCMV-hCGPx for cotransfection of 293 cells with pJM17, thereby to obtain the recombinant adenovirus AdCMV-hCGPx, Cultured ECV304 cells were transfected with AdCMV-hCGPx for 24, 48 and 72 h, respectively, with the cells transfected with the empty vector serving as control, and hCGPx gene expression was then examined in the transfected cells. The transfected cell viability and apoptotic cell ratio were evaluated after treatment of the cells with H2O2. Results The expression ratio of hCGPx gene was significantly higher in the AdCMV-hCGPx-transfected cells than in those with empty vector transfection （P〈0.01）. The hCGPx gene-transfected cells showed significantly higher viability and significantly lower apoptotic ratio than the control cells following challenge with H2O2-induced oxidative damage. Conclusion hCGPx gene transfer mediated by recombinant adenovirus protects the vascular endothelial cells fi＇om oxidative damage in vitro, possibly due to the antioxidative and apoptosis-inhibiting effect of hCGPx.