血管紧张素转换酶2基因转染对人血管内皮细胞中氧化应激水平的影响

Effects of Recombinant Angiotensin-converting enzyme 2 Gene on Oxidative Stress Levels in Human Endothelial Cells

背景血管紧张素转换酶2(ACE2)是迄今为止发现的人类 ACE 的第一个同源基因,是血管紧张素Ⅱ(AngⅡ)的主要降解酶,但其在氧化应激中的调控作用尚不清楚。目的探讨重组 ACE2基因转染对体外培养人血管内皮细胞中由 AngⅡ诱导的 NAPDH 氧化酶 p22^(phox)表达和丙二醛(MDA)水平的影响。方法克隆和构建含人 ACE2基因全长的重组质粒(pACE2),并将之转染人人血管内皮细胞中。分别采用实时定量 PCR 和 Western印迹技术检测转染细胞中的 p22^(phox)的 mRNA 与蛋白表达情况。采用硫代巴比妥酸比色法测定细胞中 MDA 含量。结果 AngⅡ(100 nmol/L)和 AngⅣ(100 nmol/L)刺激后均可诱导人内皮细胞中 p22^(phox)的 mRNA 及蛋白表达大大增加,伴有细胞中 MDA 水平升高(n=6,P 均<0.01)。pACE2基因转染可抑制细胞中由 AngⅡ和 AngⅣ诱导的 p22^(phox)表达,同时伴 MDA 水平下调(n=5～6,P 均<0.01)。结论 ACE2基因过表达可明显抑制人内皮细胞中 p22^(phox)的表达,而且降低 MDA 水平,提示 ACE2具有一定的抗氧化效应。通过调节 ACE2基因活性和表达,很可能成为氧化应激相关疾病如高血压病防治的重要手段。

Backgroup and objective Angiotensin-converting enzyme 2 （ACE2） is the first known homologue of human ACE gene up till now, however, its regulatory role in oxidative stress remains unclear. Our aim is to in- vestigate the effects of recombinant ACE2 gene transfer on the expression of p22^phox , a key subunit of NADPH oxidase, and malonyldialdehyde （MDA） levels induced by angiotension（Ang） Ⅱ in cultured human endothelial cells. Methods A recombinant plasmid encompassing human ACE2 gene （pACE2） was constructed and transfected into these cells. The mRNA and protein levels of p22^phox in endothelial cells were determined by real-time PCR and Western blotting, respectively. MDA contents were measured by thiobarbiturie acid colorimetric method in cells. Results The mRNA and protein expressions of p22^phox were drastically enhanced after exposures of endothelial cells to Ang Ⅱ（100 nmol/L） and Ang Ⅳ （100 nmol/L）, accompanied by an increase in MDA contents （n=6, P〈0.01, respectively）. Concomitantly, a significant downregulations of p22^phox expression and MDA content was shown in endothelial cells pretreated with pACE2 gene transfer （n=5-6, P〈0.01, respectively）. Conclusion ACE2 gene overexpression diminished p22^phox expression and MDA content in endothelial cells induced by Ang Ⅱ, suggesting that ACE2 has antioxidative properties to some extent and may be a novel therapeutic target for the oxidative stress-related diseases such as essential hypertension.