RNA干扰技术选择性下调大鼠血管紧张素Ⅱ 1a型受体及其作用的实验研究

Selective knockdown of Angiotensin Ⅱ receptor subtype 1a in rat vascular smooth muscle cells by RNA interference

目的用RNA干扰技术选择性下调大鼠血管平滑肌细胞(VSMCs)上血管紧张素Ⅱ1a(AT1a)型受体的表达,并检验其对细胞活性及增殖的影响。方法构建携带U6启动子和AT1a特异短发夹RNA(shRNA)编码序列的质粒载体pAT1a-shRNA1,pAT1a-shRNA2,以含非特异性shRNA编码序列的质粒载体pGenesil-Con(pCon)为对照,转染原代培养的大鼠主动脉VSMCs,通过半定量RT-PCR和Westernblot法检测AT1a、AT2受体的表达情况,以内参照β-肌动蛋白进行标化。用四甲基氮唑盐(MTT)比色法(测A490nm值)检验其对细胞活性及血管紧张素Ⅱ促增殖效应的影响。结果pAT1a-shRNA1使AT1a的mRNA和蛋白质表达分别降低82%和69%,pAT1a-shRNA2使之分别降低77%和56%,差异有统计学意义。转染对照质粒组与空白对照组AT1a受体表达差异无统计学意义;各组AT2受体表达差异无统计学意义。单纯转染质粒各组A490nm差异无统计学意义,给予血管紧张素Ⅱ刺激后,pAT1a-shRNA1、pAT1a-shRNA2组A490nm显著低于空白对照组和对照质粒组。结论RNA干扰技术能选择性下调原代培养的VSMCsAT1a受体的表达,并显著抑制血管紧张素Ⅱ促细胞增殖的效应,无明显细胞毒性。这可能为相关基因的功能研究提供新的方法,亦可能为心血管疾病基因治疗的探索开拓新的思路。

Objective To selectively knockdown the expression of Angiotensin Ⅱ receptor subtype 1a （AT1aR） in rat vascu1ar smooth muscle cells （VSMCs） by RNA interference and the sequential effects on cellu1ar viability and proliferation. Methods The primary cultured rat aortic VSMCs were transfected by p1asmids pAT1a-shRNA1 and pAT1a-shRNA2, each carrying an U6 promoter and an AT1a-specitic shRNA-coding temp1ate sequence, or by a control p1asmid pGenesil-Control （pCon） carrying a nonspecitic shRNA-coding sequence. The mRNA and protein expressions of AT1 a, AT2 were analyzed by semiquantified RT-PCR and Western blot, respectively and normalized to the internal control gene β-actin. Cellu1ar viability and proliferation were determined with methylthiazoletetrazolium （MTY） assay. Results AT1a mRNA and protein were reduced by 82% and 69% by pAT1a-shRNA1, 77% and 56% by pAT1a-shRNA2, respectively while no change was found in pCon treated VSMCs. AT2 receptor level in VSMCs remains unchanged after various treatments. The A490nm values obtained by MTT measurements were simi1ar among groups in the absence of Ang Ⅱ but decreased significantly in pAT1a-shRNA1 and pAT1a-shRNA2 treated VSMCs in the presence of Ang Ⅱ. Conclusion RNA interference can selectively knockdown AT1 a expression in cultured VSMCs and attenuate the Ang Ⅱ induced cell proliferation. Future studies axe warranted to explore the potential role of RNA interference on AT1 function and as a new gene therapy tool for cardiovascu1ar diseases.