TRPM4通道在心肌缺血再灌注损伤中的作用

发布时间：2018-11-03 17:06

【摘要】：冠心病严重威胁人类的健康，在全球每年有720万人死于冠心病。在急性心肌梗死之后迅速而确切的心肌再供血是减少心肌梗死及改善患者预后的最有效的治疗策略。近年来，随着溶栓疗法、经皮腔内冠状动脉成形术（PTCA）、冠状动脉旁路移植术（CABG），使心肌缺血后重新得到血液再灌注。但有时缺血后再灌注，不仅不能使心脏功能恢复，反而出现心肌的结构、功能障碍，甚至梗死，形成进一步损伤，既心肌缺血再灌注损伤(I/R)。而怎么减少心肌缺血再灌注损伤为本研究的重点。本研究拟通过建立大鼠的心肌缺血再灌注损伤的模型；并利用H9c2心肌细胞模拟心肌缺血再灌注损伤，分析TRPM4通道与心肌缺血再灌注的关系。方法和结果如下：方法首先利用免疫组织化学方法确定大鼠心肌组织中TRPM4通道蛋白的表达。并建立大鼠的心肌缺血再灌注损伤（I/R）模型，在I/R前给予TRPM4阻断剂9-Phe-nanthrol（9-Phe），分析9-Phe对I/R的影响。接下来利用H9c2心肌细胞研究9-Phe的心肌保护作用机制。为模拟细胞的心肌缺血再灌注损伤，将H9c2细胞暴露在H2O2中或给予缺氧/复氧（hypoxia/reoxygenation,H/R）刺激，，研究9-Phe对细胞保护作用的效果。并通过siRNA下调TRPM4的表达水平后，研究该蛋白对心肌缺血再灌注损伤的影响。结果在大鼠实验中TRPM4通道阻断剂9-Phe对心肌缺血再灌注损伤有心肌保护作用，能明显减少心肌梗死。9-Phe组明显小于对照组(分别为9.2±1.1%，37.5±7.6%; p0.01)。在细胞实验利用MTT方法对H9c2心肌细胞的成活率进行评测。9-Phe处理后将细胞暴露于浓度为200μM H2O2中4小时，与H2O2对照组或DMSO＋H2O2对照组相比有明显的细胞保护作用（吸光度分别为0.34±0.01，0.21±0.01及0.19±0.02）。同样9-Phe对经历H/R的H9c2亦有保护作用。9-Phe＋H/R，H/R，DMSO＋H/R的normalized吸光度分别为1.08±0.05，0.66±0.10和0.60±0.04。接下来运用转染的方法将TRPM4-siRNA转染入H9c2细胞中，通过real timePCR及Western blot的方法证明了TRPM4-siRNA可下调约80%的TRPM4的表达。TRPM4Knockdown的H9c2细胞对H2O2的耐受性明显增强，而9-Phe对其无明显作用。说明9-Phe对H9c2的细胞保护作用是通过抑制TRPM4通道来实现的。结论 1.TRPM4通道阻断剂9-Phe在大鼠心肌缺血再灌注损伤中减少心肌梗死。 2.9-Phe对H2O2或H/R所致的H9c2细胞损害有细胞保护作用。 3.TRPM4Knockdown对H2O2或H/R所致的H9c2细胞损害有细胞保护作用。总之9-Phe是通过抑制TRPM4通道对心肌缺血再灌注有心肌保护作用。
[Abstract]:Coronary heart disease is a serious threat to human health, 7.2 million people die of coronary heart disease every year in the world. Rapid and accurate myocardial reflow after acute myocardial infarction is the most effective treatment strategy to reduce myocardial infarction and improve the prognosis of patients. In recent years, with thrombolytic therapy, percutaneous transluminal coronary angioplasty (PTCA),) and coronary artery bypass grafting (CABG),) have been used to restore blood reperfusion after myocardial ischemia. But sometimes after ischemia reperfusion not only can not make the heart function recover but also appear myocardial structure dysfunction and even infarction and form further injury that is myocardial ischemia-reperfusion injury (I / R). How to reduce myocardial ischemia reperfusion injury is the focus of this study. The purpose of this study was to establish a model of myocardial ischemia-reperfusion injury in rats and to analyze the relationship between TRPM4 channel and myocardial ischemia-reperfusion injury by using H9c2 cardiomyocytes to simulate myocardial ischemia-reperfusion injury. Methods and the results were as follows: firstly, the expression of TRPM4 channel protein in rat myocardium was determined by immunohistochemical method. The model of myocardial ischemia-reperfusion injury (I / R) was established in rats. 9-Phe-nanthrol (9-Phe) was given before I / R to analyze the effect of 9-Phe on I / R. Then H9c2 cardiomyocytes were used to study the mechanism of myocardial protection of 9-Phe. In order to simulate myocardial ischemia-reperfusion injury, H9c2 cells were exposed to H2O2 or stimulated by hypoxia / reoxygenation (hypoxia/reoxygenation,H/R) to study the protective effect of 9-Phe on cells. After siRNA down-regulated the expression of TRPM4, the effect of the protein on myocardial ischemia-reperfusion injury was studied. Results 9-Phe, a TRPM4 channel blocker, had myocardial protective effect on myocardial ischemia-reperfusion injury in rats. The myocardial infarction was significantly reduced in the 9-Phe group than in the control group (9.2 卤1.1 vs 37.5 卤7.6, respectively). P0.01) The survival rate of H9c2 cardiomyocytes was evaluated by MTT method in cell experiment. After 9-Phe treatment, the cells were exposed to 200 渭 M H2O2 for 4 hours. Compared with H2O2 control group and DMSO+H2O2 control group, the cell protective effect was obvious (absorbance was 0.34 卤0.01 and 0.19 卤0.02, respectively). The normalized absorbance of 9-PHE + H / R + H / R / DMSO + H / R was 1.08 卤0.05 卤0.66 卤0.10 and 0.60 卤0.04, respectively. Then TRPM4-siRNA was transfected into H9c2 cells by transfection method. The results of real timePCR and Western blot showed that TRPM4-siRNA could down-regulate the expression of TRPM4 by about 80%. The tolerance of TRPM4Knockdown H9c2 cells to H2O2 was significantly enhanced. But 9-Phe had no obvious effect on it. The results suggest that the cell protection of 9-Phe against H9c2 is achieved by inhibiting the TRPM4 channel. Conclusion 9-Phe, a 1.TRPM4 channel blocker, can reduce myocardial infarction in rats with myocardial ischemia reperfusion injury. 2.9-Phe has cytoprotective effect on H9c2 cell damage induced by H2O2 or H / R. 3.TRPM4Knockdown has cytoprotective effect on H9c2 cell damage induced by H2O2 or H / R. In conclusion, 9-Phe has myocardial protective effect on myocardial ischemia reperfusion by inhibiting TRPM4 channel.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R541.4

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