过刊浏览

血管紧张素Ⅱ受体阻断对心肌成纤维细胞信号转导相关基因表达谱的影响

蒋小英, 高广道, 王新凤, 林元喜, 王亚文, 杨予白

西安交通大学医学院遗传学与分子生物学系.陕西,西安 710061；西安交通大学医学院生理学与病理生理学系.陕西,西安 710061

摘要

为了研究血管紧张素Ⅱ（angiotensinⅡ，AngⅡ）受体在成年大鼠心肌成纤维细胞的信号转导机制，分离及培养成年Sprague-Dawley大鼠心肌成纤维细胞，采用免疫组化染色测定AngⅡ受体的蛋白表达。将细胞随机分为四组进行药物干预48h：AngⅡ组、AngⅡ+losartan组、AngⅡ+PD123319组和AngⅡ+losartan+PD123319组。抽提mRNA制备cDNA探针，与G蛋白耦联受体信号通路发现者基因芯片杂交，筛选表达差异的基因。发现血管紧张素Ⅱ 1型（angiotensinⅡ type1，AT1）受体被losartan阻断后，AngⅡ刺激的心肌成纤维细胞血管紧张素Ⅱ2型（angiotensinⅡ type2，AT2）受体蛋白高表达；34个基因表达差异在2倍以上，30个下调，4个上调，其最大改变不超过20倍；9条信号通路被活化：cAMP／PKA、Ca~(2+)、PKC、PLC、MAPK、PI--3K、NO--cGMP、Rho、NF--#kappa#B通路。当AT2受体被PD123319阻断时，64个基因表达差异在2倍以上，48个下调，16个上调；11条途径基础活化，其中7个基因的改变在30倍以上：Cyp19a1（37倍）、I11r2（42倍）、Cflar（53倍）、Bcl21（31倍）、Pik3cg（278倍）、Cdknla（90倍）、Agt（162倍）。在AT1受体阻断的基础上再阻断AT2受体，46个基因表达差异在2倍以上，36个下调，10个上调；11条信号途径全部活化。其结果与单独阻断AT2受体信号途径基本一致。RT--PCR选取IL--1#beta#和TNF--#alpha#进行验证，结果与芯片各组间的变化趋势基本相符。结果表明，在成年大鼠心肌成纤维细胞，AT2受体阻断明显不同于AT1受体阻断，在信号转导通路相关基因表达谱上，两者有显著差异。

关键词： 心肌成纤维细胞; AT2受体; cDNA芯片

Alteration of signal transduction--associated gene expression in rat cardiac fibroblasts induced by blocking angiotensin Ⅱ receptors

Jiang Xiaoying, Gao Guangdao, Wang Xinfeng, Lin Yuanxi, Wang Yawen, Yang Yubai

Department of Genetics and Molecular Biology,Medical College of Xi'an Jiaotong University.Xi'an 710061,Shaanxi；China

Abstract

To investigate the molecular mechanism of angiotensin Ⅱ （Ang Ⅱ） receptor activation in adult rat cardiac fibroblasts, the expressions of cell signal transduction-associated genes were studied by using cDNA microarray. Cardiac fibroblasts of adult SpragneDawley rats （230--250 g） were isolated and cultured. The cells were divided into 4 groups： Ang Ⅱ, Ang Ⅱ + losartan, Ang Ⅱ + PD123319, Ang Ⅱ + losartan + PD123319. The expressions of Ang Ⅱ receptors were studied by immunohistocbemical staining. Total RNA was extracted and purified. After cDNA synthesis and biotin-16-dUTP labeling, the probes were denatured and hybridized with GEArray Q Series mouse G Protein-coupled Receptors Signaling Pathway Finder Gene Array （MM-025） containing 96 genes associated with 11 pathways. The arrays were scanned with a Uniscandl000 scanner and further analyzed with GEArray Analyzer software. RT-PCR was used to further confirm the results of gene microarray. The results of immunohistochemical staining showed that the expression of Ang Ⅱ type 2 （AT2） receptor was evidently induced by Ang Ⅱ stimulation when Ang II type 1 （AT1） receptor was blocked. The results of gene array indicated that blocking AT1 receptor changed 34 genes （more than 2 folds）, 30 were down-regulated and 4 were upregulated. The maximum change was not beyond 20 folds. The following 9 pathways were activated： cAMP/PKA, Ca~(2+), PKC, PLC, MAPK, PI-3 kinase, NO-cGMP, Rho, NF-#kappa#B pathways. Blockade of AT2 receptor caused 64 genes changing more than 2 folds （48 were down-regulated and 16 were up-regulated）. Eleven pathways were basically activated. The change of the following 7 genes was over 30 folds： Cypl9al （37 folds）, Il1r2 （42 folds）, Cflar （53 folds）, Bcl21 （31 folds）, Pik3cg （278 folds）, Cdkn1a （90 folds）, Agt （162 folds）. According to the activated extent, the signal transduction pathways in turn were PI-3 kinase, NF-#kappa#B and JAK-STAT pathways. Blocking both AT1 and AT2 receptors changed 46 genes more than 2 folds （36 were down-regulated and 10 were up-regulated）. Eleven pathways were basically activated. The results of RT-PCR of IL-1#beta# and TNF-#alpha# confirmed the observations in gene microarray. Our results show that Ang II can induce a high expression of AT2 receptor in adult rat cardiac fibroblasts when AT1 receptor is blocked, and the signal mechanism of AT2 receptor is clearly different from that of AT1 receptor.

Key words: cardiac myoblasts;receptor, angiotensin, type 2;cDNA microarray

收稿日期：　　录用日期：

通讯作者：　　E-mail:

引用本文：

蒋小英, 高广道, 王新凤, 林元喜, 王亚文, 杨予白. 血管紧张素Ⅱ受体阻断对心肌成纤维细胞信号转导相关基因表达谱的影响[J]. 生理学报 2006; 58 (6): .

Jiang Xiaoying, Gao Guangdao, Wang Xinfeng, Lin Yuanxi, Wang Yawen, Yang Yubai. Alteration of signal transduction--associated gene expression in rat cardiac fibroblasts induced by blocking angiotensin Ⅱ receptors. Acta Physiol Sin 2006; 58 (6): (in Chinese with English abstract).