DPP6对豚鼠心室肌细胞电活动的影响及其机制的研究

发布时间：2018-04-11 19:25

本文选题：DPP6 + 动作电位　；参考：《河北医科大学》2017年硕士论文

【摘要】：心室纤颤(ventricular fibrillation)是引起心搏骤停(cardiac arrest)和心脏猝死(sudden cardiac death)最常见的原因,通过对病人的病因学、基因学研究和动物模型研究发现其发生常常是由于心脏离子通道的异常而出现。特发性室颤是遗传性室颤的一种类型,由于其病因未知和临床症状隐蔽而具有极高的致死率。前期有研究提示一种家族特发性室颤的发生与二肽基肽酶样蛋白(Dipeptidyl peptidase-like protein,DPP)的亚型6(DPP6,又称DPPX)的表达升高密切相关,DPP6的异常高表达可引起家族特发性室颤的病理机制目前未知。DPP6是一种公认的Kv4通道调节亚基,而后者是介导心脏中瞬间外向钾电流(Ito)的主要通道,对正常心律的维持起重要作用,但目前对DPP6在心脏中的作用尤其是对心律失常发生的影响很少有报道,目前认为DPP6异常导致心脏(尤其是浦肯野纤维)Ito电流增大是导致室颤的一个主要原因。虽然DPP6是Kv4通道的调节亚基,但有研究在神经系统发现DPP6除影响A型钾电流外,对钠通道也有影响,并且发现在没有Kv4通道表达的区域也有DPP6的表达。由此我们提出一个重要的问题,DPP6是否对心脏其它的离子通道也具有调节作用进而影响室颤的发生。为回答此科学问题,本研究将在豚鼠心室肌细胞成功过表达DPP6后,通过观测过表达DPP6对豚鼠心室肌细胞动作电位以及相关离子通道(如钠通道、L型钙通道等)的影响来发现其可能的细胞电生理学机制,此研究将为揭示DPP6在某种特发性室颤的病因、病理机制、疾病的预防和治疗提供重要的实验基础,具有重要的基础研究和临床意义。目的:(1)评价腺病毒介导的DPP6过表达载体在豚鼠心室肌细胞的过表达效果(2)过表达DPP6对豚鼠心室肌细胞的动作电位的影响。(3)过表达DPP6对豚鼠心室肌离子通道(钠通道、L型钙通道、IKr、IKs)的影响及机制。方法:(1)利用荧光显微镜观测腺病毒携带的绿色荧光蛋白,实时定量PCR(qPCR)技术,蛋白免疫印迹技术来评价腺病毒介导的DPP6过表达载体在豚鼠心室肌细胞的过表达效率;(2)应用全细胞电流钳技术记录培养后的豚鼠心室肌细胞的动作电位,观察过表达DPP6对心室肌细胞动作电位的影响;(3)利用电压钳技术检测在豚鼠心室肌细胞过表达DPP6对钠通道、钙通道以及IKr、IKs电流的影响及其机制。结果:(1)感染DPP6过表达腺病毒的豚鼠心室肌细胞培养48小时后,用荧光显微镜可观测到心室肌细胞有绿色荧光蛋白的表达;通过qPCR技术检测发现DPP6 mRNA的表达水平明显提高;利用蛋白免疫印迹技术发现DPP6蛋白表达明显提高;(2)过表达DPP6的豚鼠心室肌细胞与对照相比,动作电位时程显著缩短,0相除极速率加快;(3)过表达DPP6的豚鼠心室肌细胞钠电流密度显著增加,激活曲线和失活曲线向超极化方向移动,且激活曲线移动更显著,复活曲线无显著变化;L型钙电流密度显著降低,激活曲线向去极化方向移动,失活曲线无变化;IKr和IKs无明显变化。结论:(1)我们前期构建的腺病毒介导的DPP6过表达载体能够成功感染豚鼠心室肌细胞,可用于相关研究;(2)过表达DPP6致豚鼠心室肌细胞动作电位时程显著缩短,0相除极速率加快,可能与过表达DPP6增加心室肌细胞钠电流,减小钙电流有关,以上结果提示DPP6对心肌电活动的影响可能具有一定的病理意义。
[Abstract]:Ventricular fibrillation (ventricular fibrillation) is caused by cardiac arrest (cardiac arrest) and sudden cardiac death (sudden cardiac death) the most common cause, the etiology of the patient, genetic studies and animal model studies have found that the occurrence is often caused by abnormal cardiac ion channels. Idiopathic ventricular fibrillation is a type of genetic ventricular fibrillation, the mortality rate of the unknown etiology and clinical symptoms hidden but has high. Early studies suggest that a family of idiopathic ventricular fibrillation and two dipeptidyl peptidase like protein (Dipeptidyl peptidase-like, protein, DPP) subtype 6 (DPP6, also known as DPPX) elevated expression closely related to the abnormal expression of DPP6 may be the pathological mechanism of familial idiopathic ventricular fibrillation induced by.DPP6 is currently unknown regulatory subunit of a putative Kv4 channel, the latter is mediated by transient outward potassium current (Ito) in the heart of the main pass On the road, play an important role in maintaining normal rhythm, but the DPP6 function in the heart especially few reports on the influence of arrhythmia, it is believed that DPP6 leads to abnormal heart (especially Purkinje fibers) is the result of a Ito current increases mainly due to ventricular fibrillation. Although DPP6 is Kv4 channel the regulatory subunit, but studies have found that DPP6 removal effect of A type potassium current in the nervous system, also have an impact on the sodium channel, and the expression of Kv4 in the absence of channel expression are also DPP6. So we put forward an important question, whether DPP6 on the cardiac ion channels also play a role in the regulation of the other influence of the incidence of ventricular fibrillation. In order to answer this scientific problem, this study in guinea pig ventricular myocytes successfully after overexpression of DPP6, through the observation of overexpression of DPP6 on action potential of guinea pig ventricular myocytes and related ion channels (such as sodium. Road, etc.) of L type calcium channel effect to identify the possible electrophysiological mechanism, this study will reveal the etiology, DPP6 in a kind of idiopathic ventricular fibrillation pathological mechanism, provide an important experimental basis for the prevention and treatment of disease, and has important clinical significance in basic research. Objective: (1) evaluation of adenovirus mediated expression of DPP6 vector in the effect of expression in ventricular myocytes of guinea pig (2) effect of DPP6 overexpression on action potential in guinea pig ventricular myocytes. (3) the over expression of DPP6 in guinea pig ventricular myocytes ion channel (sodium channel, L type calcium channel, IKr, IKs) effect and the mechanism. Methods: (1) carrying green fluorescent protein by fluorescence microscope observation of adenovirus, real-time quantitative PCR (qPCR), Western blotting to evaluate the adenovirus mediated expression of DPP6 vector in the expression efficiency of guinea pig ventricular myocytes; (2) using the whole cell current clamp technique Action potentials in guinea pig ventricular myocytes cultured, observed the effect of DPP6 expression on ventricular myocyte action potential; (3) detected by voltage clamp technique in guinea pig ventricular myocytes overexpressing DPP6 on sodium channel, calcium channel and IKr, the current of IKs and its mechanism. Results: (1) training 48 hours of infection in guinea pig ventricular myocytes DPP6 expression after adenovirus, using fluorescence microscopy observed the expression of ventricular myocytes with green fluorescence protein; detected by qPCR found that the expression level of DPP6 mRNA increased significantly; by protein blot technique that DPP6 protein expression increased significantly; (2) the expression of DPP6 in guinea pigs the ventricular muscle cells compared with the control group, significantly shorten the action potential, 0 phase speed rate; (3) expression in guinea pig ventricular myocytes DPP6 density increased significantly, the curves of activation and inactivation to hyperpolarization The direction of movement, and the activation curve movement is more significant, the curve had no significant change; L type calcium current density was significantly decreased, the activation curve in the depolarizing direction, the inactivation curve changes; IKr and IKs did not change significantly. Conclusion: (1) adenovirus vector we previously constructed DPP6 expression vector can guide successful infection in guinea pig ventricular myocytes, can be used in related research; (2) overexpression process significantly reduced DPP6 induced action potential of guinea pig ventricular myocytes, 0 phase speed rate, and overexpression of DPP6 increased ventricular myocytes, decrease calcium current, the above results suggest that the effect of DPP6 on myocardial electrical activity may have pathological meaning.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.75

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