MicroRNA-21对心房颤动心肌纤维化及心肌成纤维细胞增殖的调控作用机制研究

发布时间：2018-08-08 11:35

【摘要】：目的心房颤动(atrial fibrillation,AF)是临床上最常见的快速型心律失常之一,心房颤动的发生发展机制一直是国内外心血管领域的研究重点、热点,已有研究证明心肌纤维化与micro RNAs之间存在一定的相关关系。本实验旨在检测micro RNA-21及其调控蛋白Smad 7在大鼠心房颤动模型中的表达变化,micro RNA-21 mimics和micro RNA-21 inhibitors对大鼠心肌成纤维细胞活化增殖的影响,探究micro RNA-21在心房颤动心肌纤维化过程中的分子机制。方法利用皮下注射异丙肾上腺素(ISO)制备大鼠心房颤动心肌纤维化模型。随机将60只SD雄性大鼠均分为空白对照组和实验组,每组30只,其中实验组给予皮下注射ISO 5mg/kg·d、空白对照组给予等剂量生理盐水皮下注射,连续注射14天后处死大鼠,取下心脏,分别获取各组心肌组织标本。用Lipofectamine?2000Reagent向大鼠心肌成纤维细胞瞬时转染micro RNA-21 mimics和inhibitors及其阴性对照,48~72 h后检测各指标。用HE和Masson染色法测算心肌组织中的心肌纤维化程度和胶原容积分数(CVF);q RT-PCR检测各组micro RNA-21,TGF-β1和Smad 7的m RNA水平;Western blot法测定各组TGF-β1和Smad 7的表达;MTT法检测micro RNA-21 mimics和inhibitors对心肌成纤维细胞活化增殖的影响。结果HE染色和Masson染色显示实验组中大鼠心肌组织间质中出现了明显的胶原纤维增生,计算心肌间质胶原容积分数(CVF)较空白对照组明显增加;相对于正常组,大鼠心房颤动模型实验组心房组织中micro RNA-21和TGF-β1表达上调,而Smad 7表达下降。转染micro RNA-21 mimics的大鼠心肌成纤维细胞中,micro RNA-21高表达,TGF-β1表达上升,Smad 7表达下降;转染micro RNA-21mimics的心肌成纤维细胞增殖活性明显增强;转染micro RNA-21 inhibitors的大鼠心肌成纤维细胞中,得到完全相反的结果。结论皮下注射ISO能诱导大鼠心房颤动并使大鼠心肌发生明显的纤维化,心房组织中micro RNA-21的高表达可通过TGF-β1/Smad信号通路降低Smad 7的表达,从而促进心肌纤维化的发生。micro RNA-21 mimics可明显增强心肌成纤维细胞增殖活性,micro RNA-21 inhibitors则明显抑制心肌成纤维细胞增殖活性。进而表明micro RNA-21是心房颤动发生机制中的一个重要的分子信号,micro RNA-21是心肌成纤维细胞活化增殖的潜在靶向分子,有利于为干预和预防心肌纤维化的发生发展提供新思路。
[Abstract]:Objective Atrial fibrillation (atrial fibrillation AF) is one of the most common tachyarrhythmias in clinic. Studies have shown that there is a correlation between myocardial fibrosis and micro RNAs. The purpose of this study was to investigate the expression of micro RNA-21 and its regulatory protein Smad 7 in rat atrial fibrillation model. The effects of micro RNA-21 mimics and micro RNA-21 inhibitors on the activation and proliferation of rat cardiac fibroblasts were investigated. To explore the molecular mechanism of micro RNA-21 in the process of atrial fibrillation myocardial fibrosis. Methods Myocardial fibrosis model of rat atrial fibrillation was established by subcutaneous injection of isoproterenol (ISO). Sixty Sprague-Dawley male rats were randomly divided into blank control group (n = 30) and experimental group (n = 30). The experimental group was subcutaneously injected with ISO 5mg/kg d, and the blank control group was subcutaneously injected with the same dose of normal saline. After 14 days of continuous injection, the rats were killed and the heart was removed. Myocardial tissue specimens were obtained from each group. After transient transfection of micro RNA-21 mimics and inhibitors into rat myocardial fibroblasts by Lipofectamine?2000Reagent for 4872 h, the indexes were detected. Myocardial fibrosis degree and collagen volume fraction (CVF) Q RT-PCR) in myocardial tissue were measured by HE and Masson staining. Micro RNA-21 TGF- 尾 1 and Smad 7 m RNA levels were detected by micro RNA-21 TGF- 尾 1 and Smad 7. The expression of TGF- 尾 1 and Smad 7 in each group was detected by Western blot. Effects of myofibroblasts on activation and proliferation. Results HE staining and Masson staining showed that collagen fiber proliferation appeared in the interstitial tissue of rats in the experimental group. The collagen volume fraction (CVF) of myocardial interstitial tissue in experimental group was significantly higher than that in control group, and was significantly higher than that in normal group. The expression of micro RNA-21 and TGF- 尾 1 was up-regulated and the expression of Smad 7 was decreased in atrial fibrillation group. The expression of TGF- 尾 1 in rat myocardial fibroblasts transfected with micro RNA-21 mimics was increased and the expression of TGF- 尾 1 was increased. The proliferation activity of myocardial fibroblasts transfected with micro RNA-21mimics was significantly increased. In rat myocardial fibroblasts transfected with micro RNA-21 inhibitors, the expression of TGF- 尾 1 was increased, and the expression of TGF- 尾 1 was increased in rat myocardial fibroblasts transfected with micro RNA-21 inhibitors. Get the opposite result. Conclusion subcutaneous injection of ISO can induce atrial fibrillation and induce myocardial fibrosis in rats. The high expression of micro RNA-21 in atrial tissue can reduce the expression of Smad 7 through TGF- 尾 1/Smad signaling pathway. Micro RNA-21 mimics can obviously enhance the proliferation of myocardial fibroblasts. Micro RNA-21 inhibitors can obviously inhibit the proliferation of myocardial fibroblasts. It is suggested that micro RNA-21 is an important molecular signal in the pathogenesis of atrial fibrillation. Micro RNA-21 is a potential target molecule for the activation and proliferation of cardiac fibroblasts, which is helpful to provide new ideas for intervention and prevention of the occurrence and development of myocardial fibrosis.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R54

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