Objective To study the inhibition of RNA interference technology on the expression of angiotensinogen ( AGT) in rats aortic endothelial cells.Methods Building plasmids expressing AGT gene specific short hairpin RNA ( shRNA ) , make up the plasmids with liposomal transfection agent by the best ratio.After transfecting passaged 3 -5 generations rat aortic cells, detect transfection efficiency by inberted fluorescence microscope on 24 h, 48 h, 72 h.The expression of AGT mRNA was detected by RT-PCR.The protein concentration in rat endothelial cells was detected by the application of BCA protein assay kit.The expression of AGT protein was detected by Western bolt.Results ( 1 ) When the plasmids combine with liposome transfection agent TMETAFECTENE EASY+by the best ratio of 1:4, the transfection efficiency is the highest (67.25 ±2.49%) .(2) The protein contents of the blank control group, negative control group and interference group were 100%, 98.4%and 24.3%, and which in interference group was significantly lower than in the blank control group and negative control group (all P<0.05).(3)The protein concentration standard curve equation was y=0.4563x, the extracted AGT protein was 0.0456-0.9126 μg/μl.(4) The AGT protein expression after ranfection pAGT-shRNA1, pAGT-shRNA2, pAGT-shRNA3, pAGT-shRNA4 were (0.11 ±0.02),(0.12 ±0.03),(0.14 ±0.01),(0.14 ±0.08);and compared with the blank control group (0.58 ±0.07) and negative control group (0.52 ±0.04), the AGT protein in these interference groups were obviously inhibited (all P<0.05), which inhibition rates were 81.1%, 79.3%, 75.8%and 75.9%.The expression of the interference among the 4 different targeted points of plasmid carrier had no obvious difference ( all P>0.05 ) .Conclusion RNA interference can inhibit AGT expression in cultured rat aortic endothelial cells, which maybe provide a new gene therapy tool for ischemic cerebral vascular diseases.%目的 研究RNA干扰技术对大鼠主动脉内皮细胞血管紧张素原( AGT )表达的抑制. 方法 构建携带AGT基因特异短发夹RNA( shRNA)编码序列的质粒载体,与脂质体转染剂以最佳配比复合,转染传代3~5代的大鼠主动脉细胞,于转染后24 h、48 h、72 h在倒置荧光显微镜下检测转染效率,采用RT-PCR法检测AGT mRNA的表达水平,应用BCA蛋白定量试剂盒来测定大鼠内皮细胞的蛋白浓度,应用Wwestern blot法检测转染前和转染后的大鼠主动脉内皮细胞 AGT 的蛋白表达. 结果 ( 1 )当质粒与脂质体转染剂TMETAFECTENE EASY+以1:4的最佳配比复合时,转染48 h时细胞转染效率最高为(67.25 ±2.49)%. (2)空白对照组、阴性对照组与干扰组转染后的大鼠主动脉内皮细胞的AGT mRNA相对表达量分别为100%、98.4%和24.3%,且干扰组的AGT mRNA相对表达量明显低于空白对照组、阴性对照组(均P<0.05). (3)大鼠内皮细胞的蛋白浓度的标准曲线方程为y=0.4563x,所提取的大鼠主动脉内皮细胞AGT的蛋白含量为0.0456~0.9126μg/μl. (4)转染pAGT-shRNA1、pAGT-shRNA2、pAGT-shRNA3、pAGT-shRNA4的AGT蛋白表达量分别为(0.11 ±0.02)、(0.12 ±0.03)、(0.14 ±0.01)、(0.14 ±0.08);与空白对照组(0.58 ±0.07)和阴性对照组(0.52 ±0.04)相比,干扰组的AGT蛋白表达受到了明显的抑制(均P<0.05),抑制率分别为81.1%、79.3%、75.8%和75.9%. 4种不同靶位点的质粒载体所干扰的表达之间无明显差异(均P>0.05). 结论携带AGT基因特异shRNA编码序列的质粒载体pAGT-shRNA可以通过RNA技术特异性抑制大鼠主动脉内皮细胞的AGT基因的表达,为治疗缺血性脑血管疾病提供新的治疗方法.
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