N-甲基-D-天门冬氨酸受体对大鼠急性心肌梗死的影响及机制研究

发布时间：2018-07-17 07:54

【摘要】：目的：1.探讨NMDAR对大鼠急性心肌梗死的影响及其机制。2.探讨NMDA对心室肌细胞钙运作的影响。方法：第一部分：持续激活和抑制NMDAR,研究NMDAR对急性心梗大鼠室性心律失常和心梗面积的影响及其具体机制。60只SD大鼠随机分为假手术组、急性心梗组、急性心梗NMDA组(NMDA组)和急性心梗+MK801组(MK801组),分别于心梗模型制作前,连续腹腔注射生理盐水(1ml/kg)、生理盐水(1ml/kg)、NMDA (NMDAR特异性激动剂,3 mg/ml/kg)和MK-801 (NMDAR特异性抑制剂,(0.2mg/ml/kg)7天。采用结扎冠状动脉左前降支法制作急性心梗模型,记录急性期和心梗后24小时内的室性心律失常发生情况,采用TTC染色测量心梗面积。采用病理学方法检测缝隙连接蛋白43(connexin43, Cx43)的表达和凋亡小体；采用western-blot检测左心室Cx43和NMDAR1的表达量。第二部分：研究激活NMDAR对大鼠心室肌细胞钙运作的影响。急性分离大鼠心室肌细胞,用NMDA ()、NMDA+MK801 ()孵育细胞,运用激光共聚焦技术测定心室肌细胞胞浆的钙浓度、肌浆网的钙浓度、钙瞬变的幅值、自发性钙释放事件的发生率。结果：第一部分：与假手术组相比,心梗组左心室NMDAR表达量升高(1.12±0.09vs 0.54±0.06,P0.01)。与心梗组相比,MK801组室性心律失常发生率显著下降,差异有统计学意义(P0.01)。MK801组心梗面积显著下降(36.58±2.57% vs 43.72±3.11%, P0.01); Cx43分布均匀、表达量较高(1.68±0.27%vs0.74±0.15%,P0.01),凋亡小体较少(13.49±3.25%vs 32.53±7.21%,P0.01)；而左心室心肌Cx43表达均显著上调、NMDAR1表达量下降(1.12±0.09 vs0.81+0.07,P0.01)。而与心梗组相比,NMDA组室性心律失常发生率显著增加,差异有统计学意义(P0.01)。NMDA组心梗面积显著增加(54.3±1.60%vs48.62±1.51%,P0.01)；Cx43分布均匀、表达量较低(0.32±0.08%vs 0.74±0.15%,P0.01),凋亡小体较多(36.26±2.58%vs 13.49±2.25%,P0.01)；而左心室心肌Cx43表达均显著下调,NMDAR1表达量上调(P均0.01)。第二部分：与对照组相比,NMDA组的胞浆钙浓度值明显升高(239.3±7.5 nmol/L vs 128.9±9.3 nmol/L, P0.01)。钙瞬变幅值在0.5Hz (0.78±0.06 vs 0.63±0.04)和1Hz (2.94±0.13 vs 0.74±0.03)的起搏下,均显著高于对照组(P0.01)。在1Hz的起搏下,NMDA组的自发性钙释放事件显著高于对照组(P0.01)。与对照组相比,NMDA组的肌浆网钙浓度显著增加(0.5Hz:3.52±0.09 vs1.41±0.08, P0.01; 1Hz:3.76±0.11 vs 1.74±0.04, P0.01),肌浆网的钙释放分数也显著升高(0.5Hz:0.68±0.07 vs 0.42±0.05, P0.01;1Hz:0.78±0.08 vs 0.43±0.06, P0.01)。结论：1.慢性激活NMDAR通过下调Cx43、增加凋亡,进而显著增加急性心梗大鼠室性心律失常发生率和心梗面积；而抑制NMDAR可以阻止急性心梗大鼠发生室性心律失常,并减轻心梗面积。2.激活NMDAR显著增加心室肌细胞钙负荷,使钙运作紊乱,并最终使肌浆网的钙释放事件显著增多。
[Abstract]:Purpose 1. To investigate the effect and mechanism of NMDAR on acute myocardial infarction in rats. To investigate the effect of NMDA on calcium in ventricular myocytes. Methods: the first part: continuous activation and inhibition of NMDAR.The effects of NMDAR on ventricular arrhythmias and myocardial infarction area in rats with acute myocardial infarction were studied. 60 SD rats were randomly divided into sham operation group and acute myocardial infarction group. Acute myocardial infarction group (NMDAR group) and acute myocardial infarction group MK801 group (MK801 group) were injected intraperitoneally with normal saline (1ml/kg), normal saline (1ml/kg) and MK-801 (NMDAR-specific agonist 3 mg/ml/kg) and MK-801 (NMDAR-specific inhibitor, 0.2mg/ml/kg) respectively. An acute myocardial infarction model was established by ligating the left anterior descending coronary artery. The incidence of ventricular arrhythmia in acute phase and 24 hours after myocardial infarction was recorded. The area of myocardial infarction was measured by TTC staining. The expression of connexin 43 (Cx43) and apoptotic corpuscles were detected by pathological method, and the expressions of Cx43 and NMDAR1 in left ventricle were detected by western-blot. Part two: to study the effect of activation of NMDAR on calcium in rat ventricular myocytes. The rat ventricular myocytes were incubated with NMDA () and NMDA MK801 (). The plasma calcium concentration, sarcoplasmic reticulum calcium concentration, the amplitude of calcium transient and the incidence of spontaneous calcium release were measured by laser confocal technique. Results: in the first part, the expression of NMDAR in left ventricle in myocardial infarction group was higher than that in sham operation group (1.12 卤0.09vs 0.54 卤0.06 卤P0.01). The incidence of ventricular arrhythmia in MK801 group was significantly lower than that in myocardial infarction group (P0.01). The area of myocardial infarction in MK801 group was significantly decreased (36.58 卤2.57% vs 43.72 卤3.11, P0.01), the distribution of Cx43 was uniform, the expression of Cx43 was higher (1.68 卤0.27%vs0.74 卤0.15P0.01), and the apoptotic body was less (13.49 卤3.25%vs 32.53 卤7.21P0.01). However, the expression of Cx43 in left ventricular myocardium was significantly up-regulated (1.12 卤0.09 vs0.81, 0.07 vs0.81, P0.01). Compared with the myocardial infarction group, the incidence of ventricular arrhythmias increased significantly (P0.01). The area of myocardial infarction increased significantly (54.3 卤1.60%vs48.62 卤1.51 P0.01), the expression of Cx43 was uniform, the expression of Cx43 was lower (0.32 卤0.08%vs 0.74 卤0.15P0.01), and the apoptotic bodies were more (36.26 卤2.58%vs 13.49 卤2.25 P0.01). However, the expression of Cx43 in left ventricular myocardium was significantly down-regulated (P 0.01). The second part: compared with the control group, the cytosolic calcium concentration in the NMDA-treated group was significantly increased (239.3 卤7.5nmol / L vs 128.9 卤9.3nmol / L, P0.01). The amplitude of calcium transient change at 0.5 Hz (0.78 卤0.06 vs 0.63 卤0.04) and 1 Hz (2.94 卤0.13 vs 0.74 卤0.03) was significantly higher than that of the control group (P0.01). Spontaneous calcium release events in NMDA group were significantly higher than those in control group (P0.01). Compared with the control group, the sarcoplasmic reticulum calcium concentration was significantly increased (0.5 Hz: 3.52 卤0.09 vs1.41 卤0.08, P 0.01; 1Hz: 3.76 卤0.11 vs 1.74 卤0.04, P 0.01), and the Ca release fraction of the sarcoplasmic reticulum was also significantly increased (0.5 Hz: 0.68 卤0.07 vs 0.42 卤0.05, P 0.011 HzW 0.78 卤0.08 vs 0.43 卤0.06, P 0.01). Conclusion 1. Chronic activation of NMDAR increased apoptosis by down-regulating Cx43, and thus significantly increased the incidence of ventricular arrhythmia and myocardial infarction area in rats with acute myocardial infarction, while inhibition of NMDAR could prevent ventricular arrhythmia and reduce myocardial infarction area of rats with acute myocardial infarction. The activation of NMDAR significantly increased the calcium load of ventricular myocytes, caused the disturbance of calcium operation, and eventually increased the number of calcium release events in the sarcoplasmic reticulum.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R542.22

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