[含血管紧张素II 1型受体短发夹RNA的质粒对大鼠血管平滑肌细胞增生的抑制作用]

[Inhibition of hyperplasia of vascular smooth muscle cells in rat by the plasmid containing the short hairpin RNA of angiotensin II type 1 receptor].

作者信息

Sun Cheng-Lin, Duan Zhi-Quan, Xin Shi-Jie, Feng Zong-Cheng, Zhang Jing-Hong, Gu Jun, Zhang Qiang

机构信息

Department of Vascular Surgery, The First Hospital Affiliated to China Medical University, Shenyang 110001, China.

出版信息

Zhonghua Wai Ke Za Zhi. 2004 Nov 22;42(22):1357-62.

PMID:15634403

Abstract

OBJECTIVE

RNA interference is a new technology that inhibit effectively the expression the specific genes. The current study was designed to investigate whether the plasmid containing the short hairpin RNA (shRNA) of angiotensin II type 1 receptor (AT(1)R) can inhibit the hyperplasia of vascular smooth muscle cells in rat.

METHODS

The plasmids containing the shRNA of AT(1)R were constructed, and transfected vascular smooth muscle cell (VSMC) to detect the effect on the AT(1)R expression by RT-PCR and Western blot, observe the shape of VSMCs by the inverted phase contrast microscope, and detect the hyperplasia of VSMCs by trypan blues staining and MTT.

RESULTS

The plasmids was certified to be in the right rank. After transfecting cells, there was significant difference (P < 0.01) in the expression of AT(1)R mRNA between the plasmid transfected group (pAT(1)R-shRNA(1) 1.37 +/- 0.15; pAT(1)R-shRNA(2) 1.45 +/- 0.12) and the control group (2.09 +/- 0.26), and there was significant difference (P < 0.01) in the expression of AT(1)R protein between the gene transfected group (pAT(1)R-shRNA1 1.12 +/- 0.04; pAT(1)R-shRNA2 1.20 +/- 0.07) and the control group (3.17 +/- 0.21). It is shown that pAT(1)R-shRNA can decrease the expression of AT(1)R mRNA and protein. There was significant difference (P < 0.01) in the Cell number between the plasmid transfected adding AngII group (pAT(1)R-shRNA1 5.48 +/- 0.44; pAT(1)R-shRNA2 5.55 +/- 0.45) and the AngII control group (8.13 +/- 0.41); there was significant difference (P < 0.01) in the Ratio of light density by MTT between the plasmid transfected adding AngII group (pAT(1)R-shRNA1 0.365 +/- 0.024; pAT(1)R-shRNA2 0.307 +/- 0.025) and the control group (0.485 +/- 0.011); It is shown that that pAT(1)R-shRNA can inhibit the hyperplasia of VSMCs, and matching the result of morphology observation.

CONCLUSIONS

The plasmids containing the shRNA of AT(1)R can inhibit the expression of AT(1)R mRNA and protein in VSMCs, and inhibit the hyperplasia of VSMCs induced by AngII in rat.

摘要

目的

RNA干扰是一种有效抑制特定基因表达的新技术。本研究旨在探讨含血管紧张素II 1型受体（AT(1)R）短发夹RNA（shRNA）的质粒是否能抑制大鼠血管平滑肌细胞的增生。

方法

构建含AT(1)R shRNA的质粒，并转染血管平滑肌细胞（VSMC），通过RT-PCR和蛋白质印迹法检测其对AT(1)R表达的影响，用倒置相差显微镜观察VSMC的形态，用台盼蓝染色和MTT法检测VSMC的增生情况。

结果

证实质粒构建正确。转染细胞后，质粒转染组（pAT(1)R-shRNA(1) 1.37±0.15；pAT(1)R-shRNA(2) 1.45±0.12）与对照组（2.09±0.26）之间AT(1)R mRNA表达有显著差异（P<0.01），基因转染组（pAT(1)R-shRNA1 1.12±0.04；pAT(1)R-shRNA2 1.20±0.07）与对照组（3.17±0.21）之间AT(1)R蛋白表达有显著差异（P<0.01）。表明pAT(1)R-shRNA可降低AT(1)R mRNA和蛋白的表达。质粒转染加AngII组（pAT(1)R-shRNA1 5.48±0.44；pAT(1)R-shRNA2 5.55±0.45）与AngII对照组（8.13±0.41）之间细胞数量有显著差异（P<0.01）；质粒转染加AngII组（pAT(1)R-shRNA1 0.365±0.024；pAT(1)R-shRNA2 0.307±0.025）与对照组（0.485±0.011）之间MTT光密度比值有显著差异（P<0.01）；表明pAT(1)R-shRNA可抑制VSMC的增生，与形态学观察结果相符。