转染血管紧张素Ⅱ1型受体反义核苷酸对血管外膜成纤维细胞增殖的影响
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四川省科委资助项目(2000145)


The Effect of Transfected AngiotensinⅡ Receptor TypeⅠAnti-Sense Nucleotide on the Proliferation of Cultured Rat Adventitial Fibroblasts
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    为了评价转染血管紧张素Ⅱ1型受体反义核苷酸对血管外膜成纤维细胞血管紧张素Ⅱ受体亚型mRNA表达,及细胞内核酸蛋白质的合成水平的作用。采用逆转录—聚合酶链反应克隆血管紧张素Ⅱ1型受体cDNA序列(476bp) ,将克隆cDNA反向插入PLXSN ,构建一完整的含血管紧张素Ⅱ1型受体的质粒,并转染入培养的血管外膜成纤维细胞,逆转录—聚合酶链反应和免疫印迹鉴定其转染后血管紧张素Ⅱ1型受体mRNA和蛋白表达。比较血管紧张素Ⅱ10 7mol/L刺激2 4h后的转染及非转染的血管外膜成纤维细胞血管素Ⅱ受体各亚型mRNA表达、细胞内核酸蛋白质的合成水平。转染组血管紧张素Ⅱ1型受体mRNA和蛋白表达量显著减少,对照组相比差异显著(P<0 .0 1)。血管紧张素Ⅱ10 7mol/L刺激2 4h后,与对照组血管外膜成纤维细胞相比,转染组血管紧张素Ⅱ1型受体mRNA表达明显减少,血管紧张素Ⅱ受体Ⅱ型mRNA表达明显增加(P<0 .0 1) ,但两组间核酸和蛋白合成均无显著差异(P>0 .0 5 )。提示反义核苷酸封闭后,血管外膜成纤维细胞血管紧张素Ⅱ1型受体mRNA表达显著抑制,同时血管紧张素Ⅱ受体Ⅱ型mRNA上调。单纯封闭血管紧张素Ⅱ1型受体mRNA并不能有效阻断血管紧张素Ⅱ介导的血管外膜成纤维细胞生长。

    Abstract:

    Aim The purpose of the study is to evaluate the role of AT-1 anti-sense nucleotide (AT-1A) in expression of subtypes of angiotensin Ⅱ(AngⅡ) receptor mRNA, and syntheses of protein and nucleic acid in cultured rat adventitial fibroblasts. Methods AT-1 cDNA sequence (476 bp) was cloned with RT-PCR and inserted into PLXSN reversely to construct an intact plasmid containing AT-1A(PAT-1A). It was transfected into cultured rat adventitial fibroblasts,and identified by RT-PCR and Western blot. Syntheses of protein and nucleic acid by 3 H-Leu and 3 H-TdR incorporation, mRNA expressions of AT-1 and AT-2 by RT-PCR, were compared between transfected and nontransfected cultured rat adventitial fibroblasts after being stimulated for 24 h by AngⅡ 10 -7 mol/L. Results AT-1 mRNA and protein were expressed significantly less in cultured rat adventitial fibroblasts than that in control (P<0.01). AT-1 mRNA expression was markedly decreased,and AT-2 mRNA obviously increased (P<0.01); but no apparent difference was found in 3 H-Leu and 3 H-TdR incorporation between transfected and nontransfected cultured rat adventitial fibroblasts after stimulation for 24h of AngⅡ10 -7 mol/L (P>0.05). Conclusion After being blocked by AT1A, expression of AT-1 mRNA in cultured rat adventitial fibroblasts was markedly suppressed,while AT-2 mRNA up-regulated at the same time. It is shown that both syntheses of protein and nucleic acid in cultured rat adventitial fibroblasts mediated by AngⅡ could not be effectively interrupted simply by AT-1A blocking.

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周兴文,杨永健,张鑫,朱峻,李刚.转染血管紧张素Ⅱ1型受体反义核苷酸对血管外膜成纤维细胞增殖的影响[J].中国动脉硬化杂志,2001,9(4):294~297.

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  • 收稿日期:2001-04-02
  • 最后修改日期:2001-07-29
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