摘要
目的 评价转染AT1反义核苷酸 (AT1A)对心肌细胞血管紧张素Ⅱ (AngⅡ )受体亚型mRNA表达 ,及蛋白质和核酸合成的作用。方法 RT PCR克隆AT1cDNA序列 (476bp) ,将克隆的AT1cDNA反向插入PcDNA3 .1(5 .4kb) ,构建一完整的含AT1A的质粒 (PAT1A) ,并转染入培养的心肌细胞 ,RT PCR和免疫印迹鉴定其转染后AT1mRNA和蛋白表达。比较AngⅡ 10 -7mol/L剌激 2 4h后的转染及非转染的心肌细胞AT1及AT2 mRNA表达 (RT PCR)、蛋白质和核酸合成 (3 H Leu及3 H TdR掺入量 )。结果 成功构建PAT1A。转染心肌细胞AT1mRNA和蛋白表达量显著减少 ,与正常对照心肌细胞相比差异有非常显著性 (P <0 .0 1)。AngⅡ 10 -7mol/L剌激 2 4h后 ,与非转染心肌细胞相比 ,转染组AT1mRNA表达明显减少 ,AT2 mRNA表达明显增加 (P <0 .0 1) ,但两组间3 H Leu及3 H TdR掺入量差异均无显著性 (P >0 .0 5 )。结论 经AT1A封闭后 ,心肌细胞AT1mRNA表达显著抑制 ,同时AT2 mRNA上调。
Objective To evaluate the role of transfected angiotensinⅡ(Ang Ⅱ) receptor AT 1 anti-sense nucleotide (AT 1A) in the expression of subtypes of AngⅡ receptor mRNA, synthesis of protein and nucleic acid in cardiomyocytes. Methods AT 1 cDNA sequence (476 bp) was cloned with RT-PCR and reversely inserted into PcDNA3.1 (5.4 kb) to construct an intact plasmid containing AT 1A (PAT 1A). The plasmid was then transfected into the cultured cardiomyocytes and identified with RT-PCR and Western blot. The synthesis of protein and nucleic acid identified by 3H-Leu and 3H-TdR incorporation, and expressions of AT 1 and AT 2 mRNA by RT-PCR, were compared between transfected and nontransfected cardiomyocytes after being stimulated with 10 -7 mol/L AngⅡ for 24 h. Results The plasmid PAT 1A were successfully constructed. The AT 1 mRNA and its protein were expressed significantly less in the transfected cardiomyocytes than in the control (P<0.01). In the transfected cardiomyocytes, AT 1 mRNA expression was markedly decreased, but that of AT 2 mRNA obviously increased (P<0.01) when compared with the nontransfected cardiomyocytes after stimulation for 24 h with AngⅡ 10 -7 mol/L; no significant difference was found in 3H-Leu and 3H-TdR incorporation between them. Conclusion After the cardiomyocytes was tranfected with AT 1A, the expression of AT 1 mRNA was markedly suppressed,while AT 2 mRNA up-regulated at the same time. Our results indicate that AT 1A blocking can not effectively interrupt the Ang Ⅱ-induced synthesis of the protein and nucleic acid in cardiomyocytes.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2001年第4期401-403,共3页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目!(3 9770 3 1 4
3 972 50 1 3 )
重庆市科委资助项目
