【摘要】：冠心病嚴(yán)重威脅人類的健康，在全球每年有720萬人死于冠心病。在急性心肌梗死之后迅速而確切的心肌再供血是減少心肌梗死及改善患者預(yù)后的最有效的治療策略。近年來，隨著溶栓療法、經(jīng)皮腔內(nèi)冠狀動(dòng)脈成形術(shù)（PTCA）、冠狀動(dòng)脈旁路移植術(shù)（CABG），使心肌缺血后重新得到血液再灌注。但有時(shí)缺血后再灌注，不僅不能使心臟功能恢復(fù)，反而出現(xiàn)心肌的結(jié)構(gòu)、功能障礙，甚至梗死，形成進(jìn)一步損傷，既心肌缺血再灌注損傷(I/R)。而怎么減少心肌缺血再灌注損傷為本研究的重點(diǎn)。本研究擬通過建立大鼠的心肌缺血再灌注損傷的模型；并利用H9c2心肌細(xì)胞模擬心肌缺血再灌注損傷，分析TRPM4通道與心肌缺血再灌注的關(guān)系。方法和結(jié)果如下： 方法 首先利用免疫組織化學(xué)方法確定大鼠心肌組織中TRPM4通道蛋白的表達(dá)。并建立大鼠的心肌缺血再灌注損傷（I/R）模型，在I/R前給予TRPM4阻斷劑9-Phe-nanthrol（9-Phe），分析9-Phe對I/R的影響。接下來利用H9c2心肌細(xì)胞研究9-Phe的心肌保護(hù)作用機(jī)制。為模擬細(xì)胞的心肌缺血再灌注損傷，將H9c2細(xì)胞暴露在H2O2中或給予缺氧/復(fù)氧（hypoxia/reoxygenation,H/R）刺激，，研究9-Phe對細(xì)胞保護(hù)作用的效果。并通過siRNA下調(diào)TRPM4的表達(dá)水平后，研究該蛋白對心肌缺血再灌注損傷的影響。 結(jié)果 在大鼠實(shí)驗(yàn)中TRPM4通道阻斷劑9-Phe對心肌缺血再灌注損傷有心肌保護(hù)作用，能明顯減少心肌梗死。9-Phe組明顯小于對照組(分別為9.2±1.1%，37.5±7.6%; p0.01)。在細(xì)胞實(shí)驗(yàn)利用MTT方法對H9c2心肌細(xì)胞的成活率進(jìn)行評(píng)測。9-Phe處理后將細(xì)胞暴露于濃度為200μM H2O2中4小時(shí)，與H2O2對照組或DMSO＋H2O2對照組相比有明顯的細(xì)胞保護(hù)作用（吸光度分別為0.34±0.01，0.21±0.01及0.19±0.02）。同樣9-Phe對經(jīng)歷H/R的H9c2亦有保護(hù)作用。9-Phe＋H/R，H/R，DMSO＋H/R的normalized吸光度分別為1.08±0.05，0.66±0.10和0.60±0.04。接下來運(yùn)用轉(zhuǎn)染的方法將TRPM4-siRNA轉(zhuǎn)染入H9c2細(xì)胞中，通過real timePCR及Western blot的方法證明了TRPM4-siRNA可下調(diào)約80%的TRPM4的表達(dá)。TRPM4Knockdown的H9c2細(xì)胞對H2O2的耐受性明顯增強(qiáng)，而9-Phe對其無明顯作用。說明9-Phe對H9c2的細(xì)胞保護(hù)作用是通過抑制TRPM4通道來實(shí)現(xiàn)的。 結(jié)論 1.TRPM4通道阻斷劑9-Phe在大鼠心肌缺血再灌注損傷中減少心肌梗死。 2.9-Phe對H2O2或H/R所致的H9c2細(xì)胞損害有細(xì)胞保護(hù)作用。 3.TRPM4Knockdown對H2O2或H/R所致的H9c2細(xì)胞損害有細(xì)胞保護(hù)作用�？傊�9-Phe是通過抑制TRPM4通道對心肌缺血再灌注有心肌保護(hù)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 鹵1.1 vs 37.5 鹵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 鹵0.01 and 0.19 鹵0.02, respectively). The normalized absorbance of 9-PHE + H / R + H / R / DMSO + H / R was 1.08 鹵0.05 鹵0.66 鹵0.10 and 0.60 鹵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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R541.4

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