遗传 ›› 2008, Vol. 30 ›› Issue (9): 1175-1181.doi: 10.3724/SP.J.1005.2008.01175

• 研究报告 • 上一篇    下一篇

siRNA靶向沉默p22phox表达对内皮细胞衰老抑制作用的研究

李虹1; 白小涓2; 刘强3; 王宁夫1

  

  1. 1. 杭州市第一人民医院南京医科大学附属杭州医院, 杭州 310006;
    2. 中国医科大学附属第一医院, 沈阳 110001;
    3. 浙江大学医学院第二附属医院, 杭州 310009

  • 收稿日期:2007-12-08 修回日期:2008-07-14 出版日期:2008-09-10 发布日期:2008-09-10
  • 通讯作者: 李虹

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

LI Hong1; BAI Xiao-Juan2; LIU Qiang3; WANG Ning-Fu1   

  1. 1. Department of Cardiology, the Affiliated Hangzhou Hospital, Nanjing Medical University, Hangzhou 310006, China;
    2. Clinical Circulatory Section of the First Affiliated Hospital in CMU, Shenyang, 110001, China;
    3. The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China
  • Received:2007-12-08 Revised:2008-07-14 Online:2008-09-10 Published:2008-09-10
  • Contact: LI Hong

摘要:

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24~36 h; 给予AngⅡ后, b-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。

关键词: 血管紧张素Ⅱ, p22phox, 衰老, siRNA, 内皮细胞

Abstract:

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, angiotensinⅡ(AngⅡ) group, siRNA group, and AngⅡ+siRNA group. Cell aging was identified by b-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 b-gal were increased in ECV-304 stimulated with AngⅡ, and p22phox mRNA and protein expression were increased in aging ECV-304 stimulated with AngⅡ, which had lower NO and higher ROS. Compared with AngⅡgroup, 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, AngⅡcould induce ECV-304 aging cultured in vitro, and the possible pathway of endothelial cell aging is that AngⅡupregulates p22phox expression, and then enhances the cell ROS level.