hOGG1重组腺病毒载体在人视网膜色素上皮细胞的表达及其抗氧化应激作用研究

The expression of recombinant adenovirus-hOGG1 vector in human retinal pigment epithelial cells and its anti-oxidative effect

目的:观察重组腺病毒pAd-CMV5-DEST-hogg1转染人视网膜色素上皮细胞ARPE-19后,高表达hOGG1对视网膜色素上皮细胞在H2O2诱导的氧化损伤中的作用。方法:鉴定和包装重组腺病毒pAd-CMV5-DEST-hogg1和腺病毒载体对照p Ad/CMV/V5-GW/lac Z,并分别转染ARPE-19细胞。H2O2(100μmol·L-1,2 h)干预转染重组腺病毒的ARPE-19细胞(RPE-Ad-hogg1)、转染腺病毒载体对照的ARPE-19细胞(RPE-Ad-lac Z)以及未转染病毒的ARPE-19细胞。蛋白免疫印迹法检测3组细胞hOGG1的表达量;流式细胞仪检测细胞内ROS的生成量;免疫细胞化学方法检测氧化损伤标志性终产物8-羟基鸟嘌呤(8-oxod G)的形成量。结果:重组腺病毒p Ad-CMV5-DEST-hogg1包装成功,并在ARPE-19细胞中表达;高表达h OGG1的视网膜色素上皮细胞较两对照组细胞显示出更少的ROS生成量(P<0.05)、更低的8-oxod G氧化损伤程度(P<0.01)。结论:视网膜色素上皮细胞hOGGl蛋白高表达,可提高ARPE-19细胞碱基水平的修复能力。

Objective：To study the effect of hOGG1 on human retinal pigment epithelial cells in H2 O2 induced oxidative stress, by observing pAd-CMV5-DEST-hogg1 transfecting APRE-19.Methods： Construct recombinant adenovirus pAd-CMV5-DEST-hogg1, control adenovirus vector pAd/CMV/V5-GW/lacZ, and transfect them into ARPE-19 cells（RPE-Ad-hogg1, RPE-Ad-lacZ） respectively.The two cell lines,together with a normal ARPE-19 cell strain, were treated by H2 O2 （100μmol· L-1 ,2 h） .The oxygen free radicals （ ROS） production was detected by Flow Cytometry（FCM）.Immunohistochemisty assessed 8-oxoguanine（8-oxodG）,which is the oxidative damage end products.Results：The construction and packaging of recombinant adenovirus-hogg1 vector were successful, and it could be expressed in ARPE-19 cells.ARPE-19 cells with high hOGG1 expression had a lower ROS and 8-oxodG production（P〈0.05） and lower degree of oxidational damage on 8-oxodG.Conclusion：A high expression of hOGG1 in ARPE-19 cells can reduce the degree of oxidative stress induced by H2 O2 .