RNAi特异性抑制A20基因对H_2O_2诱导人脐动脉血管平滑肌细胞增殖的影响  

Effects of RNA interference-inhibited A20 gene on H_2O_2-induced proliferation of human umbilical artery smooth muscle cells

作　　者：李蕾[1] 高静[1] 陈海英[1] 俞帅[1] 曲鹏[1] 

机构地区：[1]大连医科大学附属第二医院心内科,辽宁省大连市116027

出　　处：《中国组织工程研究与临床康复》2009年第24期4658-4662,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30670836)~~

摘　　要：背景:锌指蛋白A20是近年来细胞内源性保护机制研究的新靶点,目前研究表明锌指蛋白A20抑制肿瘤坏死因子,脂多糖等多种炎性细胞因子诱导核因子κB信号通路激活所引起的血管平滑肌细胞增殖,但有关A20抗细胞氧化损伤方面的影响报道甚少。目的:观察锌指蛋白A20基因干扰对H2O2诱导的人脐动脉血管平滑肌细胞增殖和细胞核中核因子κB表达的影响及可能的分子生物学机制。设计、时间及地点:随机对照实验,于2007-03/2008-06在大连医科大学附属第二医院中心实验室完成。材料:体外培养人脐动脉血管平滑肌细胞,构建针对Genbank中人A20的基因序列,采用脂质体LipofectamineTM2000介导80pmolsiRNA,6μL转染。方法:将培养的第4代人脐动脉血管平滑肌细胞随机分6组,①空白组:培养基培养,未给与任何干预。②A20siRNA组:转染A20 siRNA 24h。③scramblesiRNA组:转染阴性对照scramblesiRNA24h。④H2O2组:H2O2持续刺激6h。⑤H2O2+A20siRNA组:转染A20siRNA24h后H2O2持续刺激6h。⑥H2O2+scramblesiRNA组:转染阴性对照scramblesiRNA24h后H2O2持续刺激6h。主要观察指标:以RT-PCR,Western-blotting检测A20基因表达变化,以四甲基偶氮唑蓝方法测定细胞增殖活性,以流式细胞技术观察血管平滑肌细胞细胞周期的变化,以Western-blotting检测细胞核中核因子κBp65的蛋白含量表达。结果:H2O2组细胞增殖率明显高于空白组,但低于H2O2+A20siRNA组(P<0.05)。H2O2组S期、G2/M期构成百分比,增殖指数均较空白组明显升高,然而较H2O2+A20siRNA组明显降低(P<0.05),空白组A20mRNA与蛋白有微弱表达,A20siRNA组较空白组表达减少(P<0.05);H2O2组A20mRNA与蛋白表达明显高于空白组但显著低于H2O2+A20siRNA组(P<0.05)干扰效率大约55%。细胞核中核因子κBp65有少量表达;A20siRNA干扰后核因子κBp65含量明显增加;H2O2刺激后核因子κBp65表达含量显著增加但明显低于H2O2+A20siRNA组(P<0.05)。结论:A2BACKGROUND： Zinc finger protein A20 is a new target to endogenous protective mechanism of cells, which can inhibit human umbilical artery smooth muscle cells （HUASMC） proliferation induced by inflammatory factors such as tumor necrosis factor （TNF） lipopolysaccharide （LPS） through activating nuclear factor K B （NF- κB） signaling pathway. However, effect of A20 on anti-oxidative injury is poorly understood. OBJECTIVE： To observe the effect of RNA interference for A20 gene on the proliferation and NF- κ B expression of HUASMC induced by H202 and its possible molecular biology mechanisms. DESIGN, TIME AND SETTING： A randomized controlled experiment was finished at the Central Laboratory of the Second Affiliated Hospital of Dalian Medical University from March 2007 to June 2008. MATERIALS： HUASMC were cultured in vitro, gene order of A20 was constructed, 80 pmol siRNA was mediated by LipofectamineTM2000, and transfected with 6 μ L H2O2. METHODS： HUASMC of fourth passage was randomly divided into six groups： （1）Blank group： no intervention. （2）A2O siRNA group： transfected with A20siRNA for 24 hours. （3）Scramble group： 24 hours of scramble siRNA. （4）H2O2 group： stimulated with H2O2 for 6 hours. （5）H2O2＋A20siRNA group： transfected with A20siRNA for 24 hours, then stimulated with H2O2 for 6 hours. （6） H2O2＋scramble siRNA group： 24 hours scramble siRNA followed by stimulating with H2O2 for 6 hours. MAIN OUTCOME MEASURES： Changes of A20 was evaluated by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot analysis. The cell proliferation was determined by MTT. In addition, the cell cycle was detected by flow cytometry. At last, the expression level of NF-κB p65 protein was evaluated by Western blot. RESULTS： The cell proliferation rate of H2O2 group was obviously higher than that of blank group, but lower than that of H2O2-A20siRNA group （P 〈 0.05）. Constituent ratio of S cell cycle, G2/M cell cycle and proliferation

关 键 词：H2O2 锌指蛋白A20 基因干扰 血管平滑肌细胞增殖 核因子κB 

分 类 号：R318[医药卫生—生物医学工程]