【作者】 吴钢；

【导师】 黄从新；

【作者基本信息】 武汉大学 ， 内科学， 2004， 博士

【摘要】 缺血是临床上常见的病理生理状态，也是导致心律失常的重要原因之一，急性和长期慢性缺血都可以导致各种房性心律失常。以往的研究表明，缺血时心肌细胞缺氧、能量代谢紊乱，导致酸中毒形成，而酸中毒可以直接影响心肌细胞膜离子电流，这是缺血导致心律失常的重要机制之一。另外，缺血时，体液因子的异常，也是心律失常发生的重要原因。近年的研究发现，急性缺血时，血管紧张素Ⅱ（angiotensin Ⅱ，AngⅡ）水平升高可以直接导致各种房性和室性心律失常，尤其和心房颤动（atrial fibrillation，AF简称房颤）的发生有着密切关系；而AngⅡ对心肌细胞离子通道的影响可能是其致心律失常的主要原因。在各种房性心律失常中，AF是发生率最高的持续性心律失常。在AF的发生和维持中，离子通道电流密度和通道动力学的变化，即离子重构（ionic remodeling）起着重要的作用。 关于缺血对心肌细胞离子电流的影响，已有学者研究了模拟缺血状态下单个心肌细胞动作电位的变化以及相关离子通道的改变，并取得了一定的成果，但既往研究的对象多为心室肌，且观察的是缺血状态下某个时间点的变化，缺乏对心房肌细胞的研究和不同时段离子电流的比较；而关于AngⅡ对心房肌细胞离子通道的影响则十分罕见。对AF发生发展过程中，离子电流变化的特点，不同的阶段分别有报道，但大多基于动物实验，也缺乏不同阶段之间的相互比较。心律失常的发生、发展是一个动态的过程，不同的时段，缺血和AngⅡ对心房肌细胞离子电流的影响可能不一致；不同的阶段，AF离子重构的特征也可能有差异。所以动态观察离子通道的变化，对揭示缺血导致房性心律失常的发生机制，有着重要的意义。因此，本课题动态观察模拟缺血对心房肌细胞电生理的动态影响；观察AngⅡ在不同状态下对心房肌细胞离子电流的影响；比较观察AF演变过程中，不同阶段心房组织病理变化和离子重构的特征。旨在探讨与缺血相关的房性心律失常发生的离子机制，为房性心律失常，尤其是AF的治疗和预防提供理论基础。 本研究第一部分以兔心房肌细胞为研究对象，采用全细胞膜片钳技术，以模拟缺血灌流液灌流，分别观察灌流0min、5min、10min、20min和30min时心肌细胞动作电位（actionpotential，AP）和Na⁺电流(fast sodium inward current，Ⅰ_Na)、L型钙电流(L-type calcium current，Ⅰ_Ca,L)、瞬间外向钾电流(transient outward potassium current，Ⅰ_to)、延迟整流性钾电流（delayed rectifying potassium current，IK）、内向整流性钾电流（inward reetifying potassium eurrent，Ik，）以及ATp敏感性钾电流（灯Psensitivepotassiumeurrent，IK、P）的变化。结果发现:在模拟缺血状态下，心房肌细胞动作电位时程（aetion potential duration，ApD）的变化呈现出先短暂延长，然后持续缩短的趋势。灌流smin时，APD50和APD90均较对照时显著延长，灌流IOmin时较对照时显著缩短，ZOmin时进一步缩短，继续观察至30min，则较Zomin时无显著变化;动作电位幅度（action potential amplitude，APA）和静息膜电位（resting membrane potential，RMp）则在模拟缺血灌流smin时无显著变化，模拟缺血灌流IOmin时显著减低，继续观察至30min，无进一步减低;整个观察过程中，0期最大除极速率（Vmax）无显著变化;IN。电流密度在灌流smin时无显著变化，10min时显著减小，继续观察无进一步减小;Ica.L电流密度在灌流smin时即显著减小，10min、20min和3Omin时均进一步减小;It。电流密度在灌流smin时无显著变化，IOmin、20min时持续减小，30min时无继续减小;IK电流密度在灌流smin时显著减小，其抑制率显著大于Iea.L，lomin、Zomin时继续减小，3omin较20min时无继续减小;在整个观察过程中，IK，电流密度无显著变化;模拟缺血灌流液灌流前，未记录到IKATP，灌流smin时，记录到开放，但电流密度很小，1 Omin时显著增大，20min时进一步增大，3Omin则较ZOmin无继续增大。 本研究第二部分，采用全细胞膜片钳技术，观察了Angll对正常状态和模拟缺血两种状态下兔心房肌细胞动作电位和离子电流的影响。结果发现:正常状态下，Angll导致兔心房肌细胞APD50和APDg。显著延长， RMP显著降低，vmax显著减小，APA无显著变化;缺血状态下，Angll使心肌细胞APD50和APDgO缩短，RMP和APA显著降低，Vmax显著减小;在正常状态和模拟缺血状态下，Angll对IN。均无显著影响，均导致Ica，L电流密度显著增大而It。、IK电流密度显著减小，IK】内向电流显著减小而外向电流无显著变化;正常状态下，Angll不能使KATP开放，模拟缺血状态下，Angll使IKATP显著增大。 本研究第三部分以风湿性心脏病患者为研究对象，观察了窦性心律（Sinusthythm，SR）、阵发性AF（paroxysmal atrial fibrillation，PAF）、慢性AF（ehronic atrialfibrillatinn，CAF）等AF发生、发展过程中不同阶段心房肌组织光镜HE染色和电镜下的变化;应用膜片钳技术研究了上述各个阶段心房肌细胞动作电位和各种离子电流的变化，探讨AF演变过程中，不同阶段、不同离子重构的特征。 结果:共15例风湿性心脏病患者进入研究，SR、PAF更多还原

【Abstract】 Ischemia is one of the most common pathological manifestations that have been seen in clinical practice, and also is one of the most important reasons for arrhythmia. Both acute and chronic ischemia can lead to several kinds of atrial arrhythmia. According to studies reported before, during ischemia, cells’ hypoxia lead to acidosis. Acidosis can affect ionic current directly and thus the arrhythmia initiate. Furthermore, the abnormal of humoral factors caused by ischemia are also arrhythmogenic. Recent years’ researches showed that angiotensin II (AngII) which rise rapidly during acute ischemia could elicit some kinds of atrial or/and ventricular arrhythmias. Especially, the high level of Angll is associated with occurrence of atrial fibrillation (AF). It was reported that arrhythmogenic mechanism of Angll is due to its effects on ion channel. Among all the atrial arrhythmias, AF is one of the most common persistent tachycardia with highest incidence. Ionic remodeling, the change of ionic current density and channel dynamics, play an important role during occurrence and maintenance of AF.Some scientists have observed the changes of action potential (AP) and ion current densities under simulated ischemia that could interpret the mechanisms of ischemia related arrhythmia. But most of their study objects were ventricular myocytes and single time point effects of ischemia were reported. The different ischemic effects in various stages and the effects on atrial myocytes are unknown. At the moment, some data were reported about the effects of Angll on ventricular myocytes. But effects of Angll on ischemic cells, especially on atrial myocytes are not seen. In recent years, the electrophysiologies of AF have been investigated in animal models and in humans. From these studies, electrical remodeling of AF was reported. But most of them are based on rapid pacing animal models. Little information is available about the ionic mechanisms underlying AF in humans. The comparison of ionic remodeling in various stages is rarely mentioned. The present study therefore was to continuously observe the effects of ischemia on electrophysiology of isolated atrial myocytes, compare the effects of Angll on cardiac myocytes in normal condition and in ischemic condition. We also investigated the pathological characters of human atrial and ionic remodeling of human atrial myocytes of different time point during AF occurrence and development. The purpose of our research was to study the dynamic ionic mechanism in ischemia related atrial arrhythmia.In the first part of our research, we continuously observed the effects of simulatedischemia on rabbit cardiac myocytes using patch clamp technique of whole cell mode. We studied the changes of AP and several ion currents such as INa, Ica,L, Ito, Ik, Iki and Ikatp in time points of 0min, 5min, 10min, 20min and 30min during simulated ischemic solution perfusion. Our data showed that under simulated ischemia, the APD was transient prolonged at first, then continuously shortened. When perfuse 5min with simulated ischemic solution, APD50 and APD90 were significant prolonged compared with Omin, while at the moment of 1 Omin perfusion, APD50 and APD90 were shortened significantly. At 20min, APD50 and APD90 were shorter than at the moment of 10min. In the time point of 30min, APD was not shortened significantly than that of 20min. APA and RMP were not significant changed until 1 Omin perfusion, and reduced continuously till 20min. During the perfusion of simulated ischemia solution, Vmax was not significant changed from beginning to end. Ina current densities were not changed in 5min but reduced significantly in 10min, and from 10min to 30min, it decreased continuously. Ica,L current densities were decreased in 5min significantly and continuous decrease till 30min. Ik current densities were reduced continuously from 5min to 20min, and did not changed significantly in 30min compared with 20min. From the beginning to the end of perfusion, IKI current densities were not changed significantly. In Omin, I更多还原