微小RNA-128通过靶向NRF2调控心肌缺血再灌注损伤的研究

发布时间：2018-02-14 19:11

本文关键词： miR-128 心肌缺血再灌注损伤 Nrf2 氧化损伤 H_2O_2　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:心肌梗死是导致冠心病患者死亡的主要原因。目前,溶栓治疗或早期介入手段为缺血部位恢复血液灌流,可以有效改善心肌缺血或者坏死。但是,心肌长时间缺血再重新灌流后,会导致部分心肌细胞死亡和心脏功能的损失,即缺血再灌注损伤。MicroRNA(miRNA,miR)是一类小的非编码RNA分子。近年来的研究表明,miRNA在心肌缺血再灌注损伤中的作用日益重要。转录因子NF-E2相关因子2(Nuclear factor erythroid2-related factor 2,Nrf2)-抗氧化反应元件(antioxidant response element,ARE)是一种十分重要的内源性抗氧化应激通路。在心肌细胞中,Nrf2蛋白的表达增加,可以有效减轻心肌细胞损害,从而显著改善心脏功能。本研究主要集中于分析miR-128在预防心肌缺血再灌注损伤中的重要作用。通过探讨miR-128在心肌缺血再灌注损伤中的作用及机制,为治疗缺血性心肌细胞超微结构的损害和改善心脏功能提供新的治疗策略方法:1.收集40例急性心肌缺血以及30例健康对照患者,从肘静脉采集研究对象外周血中miR-128表达水平。2.构建大鼠心肌缺血模型。用免疫组化和实时定量PCR方法检测miR-128和Nrf2的m RNA水平。3.双荧光素酶报告基因检测Nrf2是否是miR-128的靶基因。4.分别构建高表达和低表达miR-128的腺病毒载体,并将其注射入大鼠心肌组织内。收集心肌组织,检测ROS水平、细胞凋亡情况、心脏功能改善情况(+dp/dtmax、LVSP、LVEDP)。Hoechst染色法、流式细胞术及TUNEL染色法检测大鼠心肌细胞的凋亡情况。5.透射电镜观察线粒体损伤情况。结果:1.在急性心肌缺血患者血清中miR-128表达水平升高。2.在缺血再灌注损伤的大鼠模型中,miR-128的表达水平增加,Nrf2的表达降低。3.体外研究H2O2刺激会显著诱导miR-128水平升高。通过生物信息学的分析发现,在Nrf2的3’UTR区有一个保守的结合位点。双荧光素酶报告基因分析的结果表明,miR-128可以有效抑制荧光素酶的活力。4.体内研究发现,过表达miR-128可以有效抑制Nrf2的表达,而抑制miR-128的水平可以增加心肌细胞中Nrf2的表达情况。伴随着心肌细胞中Nrf2表达水平的升高,其下游基因HO1、NQO1、SOD1水平升高。在大鼠心肌组织中抑制miR-128后,抗氧化相关酶的表达水平随之升高,而心肌组织中ROS水平明显下调。同时,抑制miR-128可以有效降低心肌细胞的凋亡并改善缺血再灌注损伤后的心肌功能。5.透射电镜分析发现,与正常对照相比,缺血/再灌注损伤组中,心肌组织中线粒体严重肿胀、呈现空泡状、心肌细胞的内外膜结构不完整。与之相反,在心肌组织中抑制miR-128以后,心肌细胞中线粒体肿胀的现象明显得到缓解。结论:1.心肌缺血的患者血清中miR-128表达水平升高。2.H_2O_2诱导原代心肌细胞miR-128表达水平的升高。miR-128通过调控Nrf2影响细胞凋亡。3.心肌缺血再灌注损伤模型中miR-128表达水平的升高,抑制miR-128影响ROS水平,减少心肌组织凋亡细胞数,从而影响心肌损伤。
[Abstract]:Objective: myocardial infarction is the leading cause of death in patients with coronary heart disease. At present, thrombolytic therapy or early intervention to restore blood perfusion at the ischemic site can effectively improve myocardial ischemia or necrosis. Myocardial ischemia and reperfusion for a long period of time will lead to the death of some myocardial cells and the loss of cardiac function. Ischemia-reperfusion injury. MicroRNA-miRNA-miRis is a class of small non-coding RNA molecules. Recent studies have shown that miRNAs play an increasingly important role in myocardial ischemia-reperfusion injury. Transcription factor NF-E2 related factor 2Nuclear factor erythroid2-related factor 2nrf2nrf2nr-antioxidant response element. The expression of nrf2 protein is increased in cardiomyocytes, which is a very important endogenous antioxidant stress pathway. This study mainly focuses on the analysis of the important role of miR-128 in preventing myocardial ischemia-reperfusion injury. By exploring the role and mechanism of miR-128 in myocardial ischemia-reperfusion injury, To provide a new therapeutic strategy for the treatment of ischemic myocardial ultrastructure damage and improve cardiac function, we collected 40 patients with acute myocardial ischemia and 30 healthy controls. The expression level of miR-128 in peripheral blood of subjects collected from cubital vein was .2.The myocardial ischemia model of rats was established. The m RNA level of miR-128 and Nrf2 was detected by immunohistochemistry and real-time quantitative PCR. The expression of Nrf2 was detected by double luciferase report gene. The target gene of miR-128. 4. Construct the adenovirus vector with high expression and low expression of miR-128, respectively. The myocardial tissue was collected, the level of ROS, apoptosis and the improvement of cardiac function were detected (dp / dtmax-LVSPP LVEDPN. Hoechst staining). Flow cytometry and TUNEL staining were used to detect apoptosis of rat cardiomyocytes. 5. Transmission electron microscopy was used to observe mitochondrial damage. Results: 1. The expression of miR-128 in serum of patients with acute myocardial ischemia was increased. The expression level of miR-128 increased and the expression of nrf2 decreased in rat model. In vitro, H2O2 stimulation could significantly increase the level of miR-128. There is a conserved binding site in the 3UTR region of Nrf2. The results of double luciferase reporter gene analysis showed that mmiR-128 could effectively inhibit the activity of luciferase. It was found that overexpression of miR-128 could effectively inhibit the expression of Nrf2 in vivo. Inhibition of miR-128 level could increase the expression of Nrf2 in cardiac myocytes. With the increase of Nrf2 expression level in cardiomyocytes, the downstream gene HO1NQO1SOD1 increased. At the same time, inhibition of miR-128 could effectively reduce the apoptosis of myocardial cells and improve myocardial function after ischemia-reperfusion injury. In the ischemia / reperfusion injury group, the mitochondria were swollen, vacuolated and the inner and outer membrane structure of the myocardial cells were incomplete. On the contrary, after inhibition of miR-128 in the myocardial tissue, the mitochondria in the ischemic / reperfusion injury group were seriously swollen and vacuolated, and the inner and outer membrane structures of the myocardial cells were incomplete. Conclusion: 1. The expression of miR-128 in serum of patients with myocardial ischemia was significantly alleviated. 2. H _ 2O _ 2 induced the increase of miR-128 expression in primary cardiomyocytes. MiR-128 affected apoptosis by regulating Nrf2. The increase of miR-128 expression in myocardial ischemia-reperfusion injury model. Inhibition of miR-128 affects the level of ROS and the number of apoptotic cells in myocardial tissue, thus affecting myocardial injury.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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