厄贝沙坦对心肌缺血大鼠室性心律失常发生及缝隙连接蛋白Cx43表达影响的研究

发布时间：2018-05-08 00:34

本文选题：厄贝沙坦 + 缝隙连接蛋白　；参考：《南昌大学》2016年博士论文

【摘要】：缺血性心脏病引起的心律失常,尤其是室性心律失常(包括室性早搏、室速和室颤)是导致患者猝死的主要原因之一。研究认为细胞内存在着大量的氧自由基、钙超载以及酸性物质,而室性心律失常是一种多种机制共同作用的过程。通过大量的研究发现,缝隙连接改变和许多病理生理变化有着联系,并且缝隙连接的改变和心律失常有着重要的联系。学者们把和心律失常有关的缝隙连接改变称为缝隙连接重构。缝隙连接是细胞间进行信息通讯以及物质交换的重要途径,对多细胞生命体有着重要的作用。心室肌细胞闰盘内缝隙连接的重要连接蛋白为Connexin 43(Cx43),很多研究者通过实验发现该物质表达与分布的异常与心律失常发生有关。因此,研究心肌缺血时Cx43表达是否异常,对缺血后发生心律失常的作用机制可提供重要的理论依据,为临床上治疗缺血后心律失常起指导作用。第一部分:厄贝沙坦作用后大鼠缺血心肌室性心律失常的发生及缝隙蛋白Cx43表达目的:探讨大鼠心肌缺血时室性心律失常的发生,并检测心肌缝隙连接蛋白Cx 43的表达。探讨大鼠心肌缺血时厄贝沙坦对心肌Cx43表达及室性心律失常发生的作用。方法:1、实验分组:随机选取40只SD大鼠,分为四组,每组10只,对照组(即假手术组:只行开胸术,而不造成心肌梗死)、陈旧性心肌梗死组、厄贝沙坦组、陈旧性心肌梗死+厄贝沙坦组。2、大鼠心肌梗死模型的构建:大鼠麻醉后,给予呼吸机辅助呼吸,在左心耳与肺动脉圆锥之间距主动脉根部3mm处结扎冠脉前降支,术后连续3天肌肉注射青霉素40万单位以预防感染。3、大鼠心梗后室性心律失常的发生次数:每周2小时监测室性心律失常的发生次数,4周末对比各组大鼠室性心律失常的发生。4、大鼠心梗后Cx43表达水平的改变:于4周末通过Western blot和免疫组化方法分别检测正常大鼠心肌与心梗大鼠心肌Cx43蛋白的表达,对比两者含量变化,阐明心肌缺血与Cx43的关系。5、ARB类药物厄贝沙坦(30mg/kg.d)灌胃干预后观察对照组大鼠与处理组大鼠,通过连续心电图监测4周,对比其室性心律失常的发生次数,阐明厄贝沙坦可否降低室性心律失常的发生。6、心肌Cx43的改变:陈旧性心肌梗死+厄贝沙坦组大鼠,给予厄贝沙坦灌胃后4周,检测Cx43表达,阐明ARB类药物对大鼠心肌Cx43蛋白表达的影响。结果:1、心梗组室速或室颤发生率与假手术组相比明显增加(P0.05)2、心梗大鼠心肌细胞磷酸化Cx43及总Cx43蛋白表达较假手术组降低(P0.05);3、对照组大鼠与厄贝沙坦处理组大鼠,通过连续心电图监测,处理组室性心律失常的发生次数明显少于对照组。4、通过Western Blot方法检测Cx43蛋白表达情况,相比于陈旧性心肌梗死组,陈旧性心肌梗死+厄贝沙坦组Cx43蛋白表达增加,差异相比较有统计学意义(P0.05)。结论:缺血可导致大鼠室性心律失常的发生明显增加,并且导致心肌细胞中Cx43的表达水平下降,使用厄贝沙坦后,陈旧性心肌梗死室性心律失常发生减少,且所致的心室缺血区Cx43含量减少的程度较陈旧性心肌梗死组减轻。第二部分:厄贝沙坦可逆转特异性低表达缝隙蛋白Cx43而增加的大鼠室性心律失常发生目的:探讨厄贝沙坦可逆转特异性低表达缝隙蛋白Cx43而增加的大鼠室性心律失常发生。方法:1、降低Cx43表达水平(Cx43基因的沉默作用)2、大鼠分为4组,包括对照组,厄贝沙坦组,注射慢病毒组即低表达组,以及注射慢病毒+厄贝沙坦。慢病毒注射至心脏边缘区。病毒注射含量为1*1011病毒数。造模结束后1)取各组心脏组织分离心肌细胞检测心肌细胞的MTT;2)取心脏组织制成切片检测心肌细胞的凋亡;3)取心脏组织进行Cx43的WB检测;4)Cx43与室性心律失常:通过连续心电图监测4周,对比Cx43表达降低大鼠与正常大鼠室性心律失常的发生次数,阐明Cx43与室性心律失常的关系。加用厄贝沙坦治疗后,观察室性心律失常发生。结果:1.通过Sh RNA慢病毒载体介导使SD大鼠心肌细胞Cx43基因沉默,从而降低Cx43表达水平。2.Cx43表达水平降低的大鼠心肌细胞较正常大鼠心肌细胞存活率,细胞活性降低,凋亡增高(P0.05)3.厄贝沙坦使得特异性低表达Cx43增加的室性心律失常发生减少。结论:Cx43基因表达降低的大鼠较正常大鼠室性心律失常的发生明显增多。缝隙蛋白数量与室性心律失常呈负相关关系。厄贝沙坦通过Cx43的升高,降低了室性心律失常的发生。
[Abstract]:Arrhythmia caused by ischemic heart disease, especially ventricular arrhythmias, including ventricular premature beat, ventricular tachycardia, and ventricular fibrillation, is one of the main causes of sudden death. Quantitative studies have found that gap junction changes are associated with many pathophysiological changes, and there is an important link between gap junction changes and arrhythmias. Scholars call the gap junction changes associated with arrhythmias known as gap junction reconstruction. Gap junctions are an important way to communicate information and material exchange among cells. Multi cell life plays an important role. The important connexion protein of gap junction in intercalated intercalary disc of ventricular myocytes is Connexin 43 (Cx43). Many researchers have found that the abnormal expression and distribution of the substance are related to the occurrence of arrhythmia by experiments. Therefore, the abnormalities of Cx43 in myocardial ischemia and arrhythmia after ischemia are studied. The mechanism can provide important theoretical basis for clinical treatment of ischemic arrhythmia. Part 1: the occurrence of ventricular arrhythmia and the expression of crevice protein Cx43 in rat ischemic myocardium after irbesartan: To explore the occurrence of ventricular arrhythmia in myocardial ischemia in rats and to detect the gap connexin Cx 43 in myocardium. To explore the effect of erbesartan on myocardial Cx43 expression and ventricular arrhythmia during myocardial ischemia in rats. Methods: 1, the experimental group: randomly selected 40 SD rats, divided into four groups, 10 in each group, and the control group (sham operation group: only thoracotomy, without myocardial infarction), old myocardial infarction group, irbesartan group, obsolete heart Muscle infarction + erbesartan group.2, rat model of myocardial infarction: after anesthesia, the rats were given ventilator assisted respiration, the anterior descending branch of the coronary artery was ligated from the left atrial appendage to the conus of the pulmonary artery from 3mm to the aorta root. 3 days after the operation, 400 thousand units of penicillin were injected to prevent the infection of.3, and the secondary ventricular arrhythmia occurred in the rats. Number: 2 hours a week, 2 hours of monitoring ventricular arrhythmia occurrence, 4 weekend compared groups of rats with ventricular arrhythmia occurrence.4, rats after myocardial infarction Cx43 expression level changes: the 4 weekend by Western blot and immunohistochemical method to detect the expression of Cx43 protein in myocardium of normal rats and myocardial infarction rats, and compare the changes of the two levels. To elucidate the relationship between myocardial ischemia and Cx43.5, the prognosis of ARB drug erbesartan (30mg/kg.d) was observed in the control group and the treatment group. Through continuous ECG monitoring for 4 weeks, the incidence of ventricular arrhythmias was compared. It was demonstrated that irbesartan could reduce the.6 of ventricular arrhythmia and the change of myocardial Cx43: old myocardium 4 weeks after irbesartan group, the expression of Cx43 was detected by irbesartan, and the effect of ARB on Cx43 protein expression in rat myocardium was examined. Results: 1, the incidence of ventricular tachycardia or ventricular fibrillation in myocardial infarction group was significantly increased (P0.05) compared with that of sham operation group (P0.05) 2, and the expression of Cx43 and total Cx43 protein in myocardial cells of myocardial infarction rats was lower than that of sham operation group. Low (P0.05); 3, the rats in the control group and the erbesartan treatment group were significantly less than the control group.4 by continuous electrocardiogram monitoring. The expression of Cx43 protein was detected by the Western Blot method. Compared to the old myocardial infarction group, the expression of Cx43 protein in the old myocardial infarction + erbesartan group was increased. The difference was statistically significant (P0.05). Conclusion: ischemia can lead to a significant increase in ventricular arrhythmia in rats and a decrease in the level of Cx43 expression in cardiac myocytes. After the use of irbesartan, old myocardial infarct ventricular arrhythmias decrease, and the decrease of Cx43 content in ventricular ischemic areas is older than that of old ones. The second part: the second part: irbesartan can reverse the specific low expression gap protein Cx43 and increase the ventricular arrhythmia in rats. It is discussed that irbesartan can reverse the specific low expression gap protein Cx43 and increase the ventricular arrhythmia in rats. Square method: 1, reduce the expression level of Cx43 (the silence of Cx43 gene) 2, the rats were divided into 4 groups, including the control group, the Irbesartan group, the injection of lentivirus group, the low expression group, and the injection of lentivirus + erbesartan. The injection of lentivirus to the marginal zone of the heart. The injection content of the virus was 1*1011 virus. After the end of the model of the model, the cardiac tissue was divided into MTT of cardiac myocytes by centrifugation muscle cells; 2) the heart tissue was obtained. Apoptosis of cardiac myocytes was detected by slicing; 3) WB detection of Cx43 in cardiac tissue; 4) Cx43 and ventricular arrhythmia: through continuous ECG monitoring for 4 weeks, Cx43 expression decreased the frequency of ventricular arrhythmia in rats and normal rats, clarified the relationship between Cx43 and ventricular arrhythmias. After the treatment with irbesartan, the ventricular heart was observed. Results: 1. the Cx43 gene of SD rat cardiomyocytes was silenced by Sh RNA lentivirus vector, which reduced the survival rate of myocardial cells in rats with lower Cx43 expression level.2.Cx43 expression level, decreased cell activity, and increased apoptosis (P0.05) 3. irbesartan increased the specific low expression of Cx43. Conclusion: the incidence of ventricular arrhythmia in rats with Cx43 gene expression was significantly higher than that of normal rats. The number of gap protein was negatively correlated with ventricular arrhythmia. The increase of erbesartan decreased the occurrence of ventricular arrhythmia by the increase of Cx43.

【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.2;R541.7

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