^*Corresponding author. Tel: +86-29-82655164; E-mail: gaogd@mail.xjtu.edu.cn

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

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¹，高广道^2,*，王新凤²，林元喜²，王亚文²，杨予白²

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

摘 要：为了研究血管紧张素Ⅱ (angiotensin II, Ang II)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Ang II受体的蛋白表达。将细胞随机分为四组进行药物干预48 h：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(angiotensin II type 1, AT1)被losartan受体阻断后，AngⅡ刺激的心肌成纤维细胞血管紧张素Ⅱ2型(angiotensin II type 2, 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：CYP19 (37倍)、IL-1R2 (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。

关键词：心肌成纤维细胞；AT2受体；cDNA 芯片

中图分类号：R363.2⁺1

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

JIANG Xiao-Ying¹, GAO Guang-Dao², WANG XIN-Feng², LI Yuan-Xi², WANG Ya-Wen², YANG Yu-Bai²

¹Department of Genetics and molecular biology, Medical college of Xi’an Jiaotong University, Xi’an 710061, China; ²Department of Physiology and Pathophysiology, Medical college of Xi’an Jiaotong University, Xi’an 710061, China

Abstract: To investigate the molecular mechanism of angiotensin II receptor activation in adult rat cardiac fibroblasts, the expression of cell signal transduction-associated genes was studied by using cDNA microarray. Cardiac fibroblasts of adult Sprague-Dawley rats (230~250 g) were isolated and cultured. The cells were divided into 4 groups: AngII, AngII + losartan, AngII + PD123319, AngII + losartan + PD123319. The expressions of AngII receptors were studied by immunohistochemical staining. Total RNA was extracted and purified. After cDNA synthesis and biotin-16-dUTP labeling, the probes were denatured and hybridized with GE Array 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 Uniscand1000 scanner and further analyzed with GE Array Analyzer software. RT-PCR was used to further confirm the results of gene microarray. The results of immunohistochemical staining showed that the expression of AT2 receptor was evidently induced by AngII stimulation when 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 up-regulated. The maximum change was not beyond 20 folds. The following 9 pathways were activated: cAMP/PKA, Ca²⁺, PKC, PLC, MAPK, PI-3 kinase, NO-cGMP, Rho, NF-κB pathways. Blockade of AT2 receptor caused that 64 genes changed 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: CYP19 (37 folds), IL-1R2 (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-κB 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-1β and TNFα confirmed the observations with gene microarray. Our results show that AngII 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