siRNA靶向沉默p22phox表达对内皮细胞衰老抑制作用的研究

The study on inhabiting endothelial cell aging by targeted silencing of p22phox

设计特异性siRNA（Short interference RNA）诱导人脐静脉内皮细胞株ECV-304细胞NAD（P）H氧化酶活性亚单位p22phox基因沉默,探讨p22phox基因沉默在血管紧张素Ⅱ（AngⅡ）诱导的ECV-304衰老中的作用及机理。应用体外转录合成3种siRNA转染体外培养的ECV-304,RT-PCR鉴定对p22phox基因沉默的效率和特异性,确立适宜的转染浓度和基因沉默的持续时间;ECV-304分为空白对照组、AngⅡ组、siRNA转染组、AngⅡ＋siRNA转染组,观察细胞衰老改变及活性氧水平,分析各组细胞p22phox的mRNA及蛋白表达。结果表明：3种siRNA中,一种对p22phox mRNA表达抑制率达到83%,在一定转染浓度范围内,siRNA诱导的基因沉默效率呈剂量依赖性,抑制效率高峰期在24-36h;给予AngⅡ后,β-gal染色阳性细胞数显著增加,出现衰老的特征性改变,衰老细胞p22phox的mRNA及蛋白表达增加,伴有一氧化氮（NO）生成减少,活性氧生成增加,siRNA诱导p22phox基因沉默后降低了活性氧水平,增加NO生成,改善了AngⅡ诱导的ECV-304细胞的衰老改变。siRNA干扰技术可成功诱导NAD（P）H氧化酶p22phox基因沉默,从而减缓AngⅡ诱导体外培养的ECV-304衰老进程,p22phox是防治衰老有希望的分子靶点。

The aim of the study was to determine the importance and possible mechanism of NAD （P）H oxidase subunits P （superscript 22phox） involved in human umbilical endothelial cell lines ECV-304 aging by special short interference RNA （siRNA）. Three siRNAs targeting p22phox were designed and synthesized in vitro, which were used to transfect ECV-304 cultured in vitro for selecting the most powerful and most suitable transfection concentration and time. The cell line ECV-304 was divided into three groups： control group, angiotensin Ⅱ （Ang Ⅱ ） group, siRNA group, and Ang Ⅱ ＋siRNA group. Cell aging was identified by β-gal stain. Reactive oxygen species （ROS） and NO level in cells and medium were measured. RT-PCR and Western blot were used to analyze mRNA and protein expression of NAD（P）H oxidase subunit p22phox. Among the 3 siRNAs, siRNA-1 was the most powerful on gene silence with 50 nmol/L transfection concentration at 24 h and 36 h. The number of positive cells stained by 13-gal were increased in ECV-304 stimulated with Ang Ⅱ, and p22phox mRNA and protein expression were increased in aging ECV-304 stimulated with AngⅡ, which had lower NO and higher ROS. Compared with Ang Ⅱ group, ROS level was decreased and NO level was increased in AngⅡ ＋siRNA group with decreased aging level. The result of the present study suggested that siRNA could induce NAD（P）H oxidase subunit p22phox gene silence, Ang Ⅱ could induce ECV-304 aging cultured in vitro, and the possible pathway of endothelial cell aging is that Ang Ⅱ upregulates p22phox expression, and then enhances the cell ROS level.