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转染血管紧张素Ⅱ受体反义核苷酸对心肌成纤维细胞的作用(PDF)

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2000年第6期
页码:
425-428
栏目:
论著
出版日期:
2000-12-01

文章信息/Info

Title:
Effect of transfecting angiotensin Ⅱ receptor anti-sense nucleotide on fibroblasts
作者:
杨永健1;  祝善俊1;  祝之明2;  胡厚祥2;  丁钢2
第三军医大学: 1. 新桥医院心内科, 2. 大坪医院野战外科研究所高血压中心, 重庆400037
Author(s):
YANG Yong-jian1;  ZHU Shan-jun1;  ZHU Zhi-ming2;  HU Hou-xiang2;  DING Gang2
1.Department of Cardiology, Xin Qiao Hospital, 2. Hypertension Center,Da Ping Hospital, Third Military Medical University, Chongqing 400037, China
关键词:
受体;  血管紧张素;  反义核苷酸;  成纤维细胞;  心肌
Keywords:
receptors;  angiotensin;  antisense nucleotide;  fibroblasts;  myocardi
分类号:
R331.38
DOI:
-
文献标识码:
A
摘要:
目的 评价转染血管紧张素 (Ang )受体 (AT1 )反义核苷酸 (AT1 A)对心肌成纤维细胞 (Fbs) Ang 受体亚型 m RNA、蛋白激酶 C(PKC)、丝裂素活化蛋白激酶 P38(P38MAPK)蛋白表达 ,及蛋白质合成的作用。方法 RT-PCR克隆 AT1 c DNA片段 (4 76 bp) ,将克隆的 AT1 c DNA片段反向插入 Pc DNA 3.1(5 .4kb) ,构建一完整的含AT1 A的质粒 (PAT1 A)。转染培养的大鼠 Fbs,RT- PCR检测转染的 Fbs AT1 m RNA表达。 Ang 10 - 7m ol/ L 刺激 2 4h后 ,比较转染及非转染的 Fas Ang 受体 AT1 及 AT2 m RNA表达 (RT- PCR)、P38MAPK和 PKC蛋白表达(western blot)、蛋白质合成 (3H- L eu掺入 )。结果 成功构建 PAT1 A。 RT- PCR显示转染 Fbs AT1 m RNA的水平降低 ,与对照 Fbs相比差异显著 (P<0 .0 1)。 Ang 10 - 7m ol/ L 刺激 2 4h后 ,与非转染 Fbs相比 ,转染Fbs AT1mRNA 明显减少,AT2mRNA 明显增加(P<0. 01) ; 但两组间PKC 和P38MAPK 蛋白表达、3H-L eu 及3H-TdR 掺入量均无显著性差异(P>0.05)。结论 经AT1A 封闭后, 能显著地抑制FbsA T1mRNA 表达, 同时上调AT1mRNA。单纯封闭AT1mRNA 并不能有效阻断AngII 介导的Fbs 蛋白质合成及Fbs 生长相关的信号转导。
Abstract:
AIM To evaluate the role of AT 1 anti sense nucleotide (AT 1A) in expressions of subtypes of angiotensinⅡ(AngⅡ) receptor mRNA,PKC,P 38 MAPK and syntheses of protein in fibroblasts(Fbs). METHODS AT 1 cDNA sequence (476 bp) was cloned with RT PCR and was inserted into PcDNA 3.1(5.4 kb) anti sensely to construct an intact plasmid containing AT 1A(PAT 1A). It was transfected into the cultured Fbs,and identified by RT PCR. Syntheses of protein (by 3H Leu incorporation), mRNA level of AT1 and AT2 (by RT-PCR ) and protein expressions of P38 MAPK and PKC (by western blot) were compared between transfected and nontransfected groups after 24 h stimulated for by AngII 10-7mol/L. RESULTS We constructed PAT1A successfully. The level of AT1mRNA decreased significantly in transfected Fbs compared with the control Fbs (P<0.01). The level of AT1mRNA was markedly decreased and that of AT2mRNA obviously increased (P<0.01) in transfected Fbs compared with nontransfected Fbs, but no apparent difference was found in 3H-Leu incorporation, expression s of PKC and P38 MAPK protein between transfected and nontransfected Fbs after 24h stimulated by AngII 10- 7 mol/L (P>0. 05). CONCLUSION After being blocked by AT1A , expression of AT1 mRNA in cultured Fbs was markedly suppressed, and AT2mRNA up-regulated at the same time. It is shown that both syntheses of protein and growth related signaling transduction in Fbs mediated.

参考文献/References

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备注/Memo

备注/Memo:
收稿日期：2000-04-17.基金项目：国家自然科学基金资助项目(39770314, 39725013)
更新日期/Last Update: 2000-12-01