血管紧张素Ⅱ1型受体的短发夹环RNA抑制大鼠血管平滑肌细胞增殖的研究  

Inhibition of hyperplasia of vascular smooth musle cells in rat by the plasmid containing the short hairpin RNA of angiotensin Ⅱ type 1 receptor

作　　者：孙成林[1] 段志泉[1] 辛世杰[1] 冯宗承[1] 张京红[1] 古峻[1] 张强[1] 

机构地区：[1]中国医科大学附属第一医院血管外科,沈阳110001

出　　处：《中华外科杂志》2004年第22期1357-1362,共6页Chinese Journal of Surgery

基　　金：国家自然科学基金资助项目 (3 0 0 0 0 163 )

摘　　要：目的　探讨血管紧张素Ⅱ 1型受体 (AT1R)的短发夹环RNA(shRNA)质粒 (pAT1R shRNA)对大鼠血管平滑肌细胞增殖变化的影响并探讨其发生机制。方法　构建pAT1R shRNA质粒转染入鼠血管平滑肌细胞中 ,应用逆转录聚合酶链反应及Westernblot检测血管平滑肌细胞 (VSMC)AT1R的mRNA和蛋白表达的变化 ;倒置相差显微镜观察VSMC形态学的变化 ;锥虫蓝染色法和MTT法检测VSMC增殖的变化等。结果　构建的质粒经测序鉴定验证与预期相符。转染细胞后逆转录聚合酶链反应的吸光度值比值结果pAT1R shRNA1和pAT1R shRNA2 组分别为 1 37± 0 15和 1 4 5± 0 12 ,与对照组 2 0 9± 0 2 6相比差异有显著性意义 (P <0 0 1) ;Westernblot吸光度值比值结果两质粒转染组分别为 1 12± 0 0 4和 1 2 0± 0 0 7,与对照组 3 17± 0 2 1相比差异有显著性意义 (P <0 0 1) ,提示两质粒均能明显降低AT1R的mRNA和蛋白表达。转染质粒同时加血管紧张素Ⅱ组的细胞计数结果两质粒转染组分别为 5 4 8± 0 4 4和 5 5 5± 0 4 5 ,与对照组 8 13± 0 4 1相比均减少、差异有显著性意义 (P <0 0 1) ,MTT法结果两质粒转染组吸光度值分别为 0 36 5± 0 0 2 4和 0 30 7± 0 0 2 5 ,与对照组 0 4 85±0 0 11相比均降低。Objective RNA interference is a new technology that inhibit effectively the expression the specific genes. The current study was designed to investigate whether the plasmid containing the short hairpin RNA(shRNA) of angiotensin Ⅱ type 1 receptor(AT 1R) can inhibit the hyperplasia of vascular smooth musle cells in rat. Methods The plasmids containing the shRNA of AT 1R were constructed, and transfected vascular smooth musle cell(VSMC) to detect the effect on the AT 1R expression by RT-PCR and Western blot, observe the shape of VSMCs by the inverted phase contrast microscope, and detect the hyperplasia of VSMCs by trypan blues taining and MTT. Results The plasmids was certified to be in the right rank. After transfecting cells, there was significant difference( P <0.01) in the expression of AT 1R mRNA between the plasmid transfected group (pAT 1R-shRNA 1 1.37±0.15; pAT 1R-shRNA 2 1.45±0.12) and the control group(2.09±0.26), and there was significant difference( P <0.01)in the expression of AT 1R protein between the gene transfected group (pAT 1R-shRNA1 1.12±0.04; pAT 1R-shRNA2 1.20±0.07) and the control group(3.17±0.21). It is shown that pAT 1R-shRNA can decrease the expression of AT 1R mRNA and protein. There was significant difference( P <0.01)in the Cell number between the plasmid transfected adding AngⅡ group (pAT 1R-shRNA1 5.48± 0.44; pAT 1R-shRNA2 5.55± 0.45) and the AngⅡcontrol group (8.13±0.41); there was significant difference( P <0.01) in the Ratio of light density by MTT between the plasmid transfected adding AngⅡ group (pAT 1R-shRNA1 0.365±0.024; pAT 1R-shRNA2 0.307±0.025) and the control group (0.485±0.011); It is shown that that pAT 1R-shRNA can inhibit the hyperplasia of VSMCs, and matching the result of morphology observation. Conclusions The plasmids containing the shRNA of AT 1R can inhibit the expression of AT 1R mRNA and protein in VSMCs, and inhibit the hyperplasia of VSMCs induced by AngⅡ in rat.

关 键 词：VSMC 质粒 对照组 短发夹环RNA 转染细胞 血管紧张素Ⅱ1型受体 血管平滑肌细胞增殖 测序 锥虫 显著性 

分 类 号：R651.12[医药卫生—外科学]