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  

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

作　　者：蒋小英[1] 高广道[2] 王新凤[2] 林元喜[2] 王亚文[2] 杨予白[2] 

机构地区：[1]西安交通大学医学院遗传学与分子生物学系,西安710061 [2]西安交通大学医学院生理学与病理生理学系,西安710061

出　　处：《生理学报》2006年第6期556-566,共11页Acta Physiologica Sinica

基　　金：This work was supported by the National Natural Science Foundation of China (No. 30271435)

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Ⅱ1型(angiotensinIItype1,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(angiotensinIItype2,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、Ca2+、PKC、PLC、MAPK、PI-3K、NO-cGMP、Rho、NF-κB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)、Il1r2(42倍)、Cflar(53倍)、Bcl21(31倍)、Pik3cg(278倍)、Cdkn1a(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-1β和TNF-α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。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-rd3 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）, Illr2 （42 folds）, Cflar （53 folds）, Bcl21 （31 folds）, Pik3cg （278 folds）, Cdknla （90 folds）, Agt （162 folds）. According to the activated extent, the signal transduction pathways in turn were PI-3 kinase, NF-rd3 and JAK-STAT pathways. Blocking both AT1 and AT2 receptors ch

关 键 词：心肌成纤维细胞 AT2受体 CDNA芯片 

分 类 号：R363[医药卫生—病理学]