腺病毒介导反义血管紧张素Ⅱ1型受体cDNA对人肺动脉平滑肌细胞生物学行为的影响  

Effects of adenoviral vector containing human angiotensin Ⅱ type 1 receptor antisense cDNA on biological action of human pulmonary artery smooth muscle cells

作　　者：涂明利[1] 王汉琴[2] 雷怀定[1] 罗国仕[1] 刘先军[1] 刘为舜[1] 熊畅[1] 刘玉全[1] 任思群[1] 

机构地区：[1]郧阳医学院附属太和医院呼吸内科,湖北十堰442000 [2]郧阳医学院解剖学教研室

出　　处：《中华结核和呼吸杂志》2005年第4期263-267,共5页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：湖北省科技攻关计划基金资助项目(2004AA304B09);湖北省教育厅资助项目(2001A39009)

摘　　要：目的探讨腺病毒介导的反义血管紧张素Ⅱ(AngⅡ)1型受体(AT1R)cDNA转染对培养的人肺动脉平滑肌细胞(PASMC)迁移、增殖和凋亡的影响。方法构建重组反义人AT1R腺病毒(AdCMVahAT1)和表达β半乳糖苷酶的对照腺病毒载体AdCMVLacZ,将培养的PASMC分为DMEM组、AdCMVLacZ组和AdCMVahAT1组,分别给予不同的干预因素干预PASMC,用半定量逆转录聚合酶链反应(RT PCR)和免疫组化及彩色图像分析法检测AT1R的表达情况,并用改良的迁移小室进行细胞迁移实验。又将培养的PASMC分为DMEM组,AngⅡ组,AdCMVLacZ+AngⅡ组和AdCMVahAT1+AngⅡ组,用流式细胞仪检测各组PASMC的增殖指数和凋亡率,绘制DNA合成直方图。结果转染AdCMVahAT1后48h的PASMC,AT1RmRNA明显低于对照组[AT1R/βactin吸光度(A)比值:AdCMVahAT1组为0.48,DMEM组为0.97,AdCMVLacZ组为0.96];AdCMVahAT1组AT1R蛋白表达水平(A值为176.39±21.63)显著低于DMEM组(A值为310.21±29.82)和AdCMVLacZ组(A值为306.45±30.09),差异均有统计学意义(P均<0.01),转染AdCMVahAT1后24h的PASMC迁移距离为(40.93±9.69)μm,显著短于DMEM组的(78.23±11.79)μm和AdCMVLacZ组的(77.80±10.66)μm,差异均有统计学意义(P均<0.01)。给予AngⅡ刺激48h,AngⅡ组PASMC的增殖指数(59.69±3.46)Objective To investigate the effect of human angiotensin Ⅱ (AngⅡ) type 1 receptor(AT_1R) antisense cDNA(ahAT_1) on migration,proliferation,and apoptosis of cultured human pulmonary artery smooth muscle cells(PASMC). Methods Two recombinant adenoviral vectors,AdCMVahAT_1 containing full length antisense cDNA targeting to human AT_1R mRNA,and AdCMVLacZ containing LacZ,were constructed by orientation clone technology and homologous recombination. The PASMC was divided into 3 groups(DMEM,AdCMVLacZ,AdCMVahAT_1) and different interventions were given to different groups. AT_1R expression was detected by RT-PCR and immunohistochemistry method; migration of PASMC was measured by Boyden′s Chamer method. Other PASMC was divided into 4 groups(DMEM,AngⅡ,AdCMVLacZ+AngⅡand AdCMVahAT_1+AngⅡ),and only the last 2 groups were respectively transfected with AdCMVLacZ and AdCMVahAT_1 before administration of AngⅡ. From 6 h to 96 h after stimulation by AngⅡ(10 -7 mol/L),proliferation index(PI) and apoptosis of PASMC were determined by flow cytometry. Results At the 48 h the level of AT_1R mRNA was significantly less in PASMC transfected AdCMVahAT_1 than that in group DMEM and in group AdCMVLacZ. The protein level showed a same difference(P<0.01). At 24 h the migration distance of PASMC also was significantly less in group AdCMVahAT_1 than that in group DMEM and Group AdCMVLacZ(P<0.01). Stimulated by AngⅡ for 48 h,in group AngⅡ the PI of PASMC markedly increased(P<0.01 vs group DMEM). But in Group AdCMVahAT_1+AngⅡ PI of PASMC clearly decreased(P<0.01 vs group AngⅡ and group DMEM respectively). There was no statistic difference of PI between group AdCMVLacZ+AngⅡ and group AngⅡ. Moreover,apoptosis peak emerged only in group AdCMVahAT_1+AngⅡ. The rate of apoptosis in those PASMC used AdCMVahAT_1 and AngⅡ was 24.70±4.04(P<0.01 vs the other 3 groups respectively). Conclusions These results indicate that AngⅡ stimulates proliferation via AT_1 receptors in human PASMC,and antisense cDNA targeting to human AT

关 键 词：血管紧张素Ⅱ1型受体 细胞生物学行为 逆转录-聚合酶链反应(RT-PCR) 介导反义 血管紧张素Ⅱ(AngⅡ) PASMC 肺动脉平滑肌细胞 DMEM 流式细胞仪检测 AT1R CDNA转染 β半乳糖苷酶 增殖指数 蛋白表达水平 AngⅡ介导 腺病毒介导 

分 类 号：R346[医药卫生—基础医学]