【摘要】：目的：1.探討NMDAR對(duì)大鼠急性心肌梗死的影響及其機(jī)制。2.探討NMDA對(duì)心室肌細(xì)胞鈣運(yùn)作的影響。方法：第一部分：持續(xù)激活和抑制NMDAR,研究NMDAR對(duì)急性心梗大鼠室性心律失常和心梗面積的影響及其具體機(jī)制。60只SD大鼠隨機(jī)分為假手術(shù)組、急性心梗組、急性心梗NMDA組(NMDA組)和急性心梗+MK801組(MK801組),分別于心梗模型制作前,連續(xù)腹腔注射生理鹽水(1ml/kg)、生理鹽水(1ml/kg)、NMDA (NMDAR特異性激動(dòng)劑,3 mg/ml/kg)和MK-801 (NMDAR特異性抑制劑,(0.2mg/ml/kg)7天。采用結(jié)扎冠狀動(dòng)脈左前降支法制作急性心梗模型,記錄急性期和心梗后24小時(shí)內(nèi)的室性心律失常發(fā)生情況,采用TTC染色測量心梗面積。采用病理學(xué)方法檢測縫隙連接蛋白43(connexin43, Cx43)的表達(dá)和凋亡小體；采用western-blot檢測左心室Cx43和NMDAR1的表達(dá)量。第二部分：研究激活NMDAR對(duì)大鼠心室肌細(xì)胞鈣運(yùn)作的影響。急性分離大鼠心室肌細(xì)胞,用NMDA ()、NMDA+MK801 ()孵育細(xì)胞,運(yùn)用激光共聚焦技術(shù)測定心室肌細(xì)胞胞漿的鈣濃度、肌漿網(wǎng)的鈣濃度、鈣瞬變的幅值、自發(fā)性鈣釋放事件的發(fā)生率。結(jié)果：第一部分：與假手術(shù)組相比,心梗組左心室NMDAR表達(dá)量升高(1.12±0.09vs 0.54±0.06,P0.01)。與心梗組相比,MK801組室性心律失常發(fā)生率顯著下降,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。MK801組心梗面積顯著下降(36.58±2.57% vs 43.72±3.11%, P0.01); Cx43分布均勻、表達(dá)量較高(1.68±0.27%vs0.74±0.15%,P0.01),凋亡小體較少(13.49±3.25%vs 32.53±7.21%,P0.01)；而左心室心肌Cx43表達(dá)均顯著上調(diào)、NMDAR1表達(dá)量下降(1.12±0.09 vs0.81+0.07,P0.01)。而與心梗組相比,NMDA組室性心律失常發(fā)生率顯著增加,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。NMDA組心梗面積顯著增加(54.3±1.60%vs48.62±1.51%,P0.01)；Cx43分布均勻、表達(dá)量較低(0.32±0.08%vs 0.74±0.15%,P0.01),凋亡小體較多(36.26±2.58%vs 13.49±2.25%,P0.01)；而左心室心肌Cx43表達(dá)均顯著下調(diào),NMDAR1表達(dá)量上調(diào)(P均0.01)。第二部分：與對(duì)照組相比,NMDA組的胞漿鈣濃度值明顯升高(239.3±7.5 nmol/L vs 128.9±9.3 nmol/L, P0.01)。鈣瞬變幅值在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,均顯著高于對(duì)照組(P0.01)。在1Hz的起搏下,NMDA組的自發(fā)性鈣釋放事件顯著高于對(duì)照組(P0.01)。與對(duì)照組相比,NMDA組的肌漿網(wǎng)鈣濃度顯著增加(0.5Hz:3.52±0.09 vs1.41±0.08, P0.01; 1Hz:3.76±0.11 vs 1.74±0.04, P0.01),肌漿網(wǎng)的鈣釋放分?jǐn)?shù)也顯著升高(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)。結(jié)論：1.慢性激活NMDAR通過下調(diào)Cx43、增加凋亡,進(jìn)而顯著增加急性心梗大鼠室性心律失常發(fā)生率和心梗面積；而抑制NMDAR可以阻止急性心梗大鼠發(fā)生室性心律失常,并減輕心梗面積。2.激活NMDAR顯著增加心室肌細(xì)胞鈣負(fù)荷,使鈣運(yùn)作紊亂,并最終使肌漿網(wǎng)的鈣釋放事件顯著增多。
[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 鹵0.09vs 0.54 鹵0.06 鹵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 鹵2.57% vs 43.72 鹵3.11, P0.01), the distribution of Cx43 was uniform, the expression of Cx43 was higher (1.68 鹵0.27%vs0.74 鹵0.15P0.01), and the apoptotic body was less (13.49 鹵3.25%vs 32.53 鹵7.21P0.01). However, the expression of Cx43 in left ventricular myocardium was significantly up-regulated (1.12 鹵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 鹵1.60%vs48.62 鹵1.51 P0.01), the expression of Cx43 was uniform, the expression of Cx43 was lower (0.32 鹵0.08%vs 0.74 鹵0.15P0.01), and the apoptotic bodies were more (36.26 鹵2.58%vs 13.49 鹵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 鹵7.5nmol / L vs 128.9 鹵9.3nmol / L, P0.01). The amplitude of calcium transient change at 0.5 Hz (0.78 鹵0.06 vs 0.63 鹵0.04) and 1 Hz (2.94 鹵0.13 vs 0.74 鹵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 鹵0.09 vs1.41 鹵0.08, P 0.01; 1Hz: 3.76 鹵0.11 vs 1.74 鹵0.04, P 0.01), and the Ca release fraction of the sarcoplasmic reticulum was also significantly increased (0.5 Hz: 0.68 鹵0.07 vs 0.42 鹵0.05, P 0.011 HzW 0.78 鹵0.08 vs 0.43 鹵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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R542.22

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