【作者】 陈琳；

【导师】 周胜华；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 研究背景心房颤动(Atrial Fibrillation, AF)是一种以心房不协调活动而导致心房机械功能恶化为特征的室上性心动过速性心律失常。房颤是一种常见的心律失常,关于房颤的触发、驱动和维持机制目前仍不是很清楚,在触发、驱动机制方面,心脏腔静脉系统如冠状静脉、上下腔静脉、肺静脉系统等部位的灶性放电可触发阵发性房颤。此外心房肌迷走神经张力异常也可能和触发机制有关,在维持机制方面,近年来,由于对房颤研究的不断深入,心房重构(atrial remodeling)越来越受到重视,心房重构包括心房电重构(AER)和心房的结构重构(AAR)。病理学上房颤分为孤立性房颤和病理性房颤。在病理性房颤的发生和维持机制中,左心房扩大被视为最重要的因素之一。心房扩大一方面促进心房内形成多折返环,另一方面改变了心房肌的电生理特性。研究表明,心律失常更多地依赖于细胞间电激动的被动传导(即被动电生理活动),并且证实细胞间缝隙连接(Gap Junction GJ)作为细胞间的一种特殊通道介导着细胞间电和化学信号的传递,在维持电和机械耦联的正常进行以及确保心肌协调收缩方面起着重要作用。组成缝隙连接的蛋白简称连接蛋白(Connexion Cx),在人体心肌中,主要有Cx31.9、Cx37、Cx40、Cx43和Cx45五种连接蛋白,其中Cx43在四个心腔均分布丰富,Cx45主要分布在心肌传导束及蒲氏纤维,Cx40主要分布在心房肌,是心房电激动传导的关键蛋白。许多基础研究认为房颤发生后在数秒至数分钟内即出现离子的浓度、离子通道的活性和磷酸化改变,数小时至数天即出现离子通道的信使核糖核酸(mRNA)表达和蛋白表达改变,包括缝隙连接蛋白(Cx)的改变。随后出现心房肌细胞肥大、凋亡、炎性细胞浸润、间质纤维化。目前对缝隙连接蛋白Cx40、Cx43变化在风湿性心脏病房颤的发生中的作用有较多的研究报道,但缝隙连接蛋白Cx40、Cx43在冠心病房颤患者左、右心房肌中的表达、分布,Cx40重构与房颤及左心房大小的关系如何还少有报道。本研究将通过对比冠心病房颤患者左右心房肌中缝隙连接蛋白Cx40、Cx43, Cx40mRNA、Cx43mRNA的表达,变化及光镜、电镜下的结构重构,分三个部分对冠心病房颤的机制进行探讨。目的：观察冠心病窦性心律正常左房大小(SR)、窦性心律左房扩大(SR+LAD)、心房颤动并左房扩大者(AF+LAD)及风湿性心脏病心房颤动并右房扩大者(AF+RAD)左、右心房肌细胞缝隙连接蛋白(Cx40mRNA、Cx40)的表达变化,探讨Cx40mRNA、Cx40在左、右心耳肌中的表达,与左、右心房大小的关系及在冠心病房颤发生中的作用。方法：选择26例接受冠状动脉旁路移植术的冠心病患者和11例接受换瓣术的风心病患者为研究对象,37例患者术前均接受冠状动脉造影检查,根据心电图和心脏彩超资料分为四组：AF+LAD组11例,AF+RAD组11例,SR+LAD组8例,SR组7例,分别于手术中切取左、右心耳标本(左心房大小正常者一般只切取右心耳肌标本)。用RT-PCR方法及免疫印迹法(Western blot)检测Cx40mRNA、Cx40的表达。结果：1. Cx40mRNA、Cx40表达量均值在扩大的右心房和正常大小的右心房肌标本中有显著差异(P<0.01)。2. Cx40mRNA、Cx40表达量均值在SR+LAD、AF+LAD组左心耳肌标本中比较无显著性差异(P>0.05)。3. Cx40mRNA、Cx40表达量均值在SR+LAD、AF+LAD和AF+RAD组左心耳肌标本中与正常大小的右心耳肌标本比较明显降低,有显著性差异(P<0.01)。结论：1.左、右心房扩大后心房肌Cx40mRNA、Cx40表达下调。2.左心房扩大并房颤后Cx40mRNA、Cx40表达无进一步下调。3.心房扩大和Cx40表达下调可能是房颤的产生和维持的重要因素。目的：观察窦性心律正常左房大小(SR)、窦性心律左房扩大(SR+LAD)、心房颤动并左房扩大者(AF+LAD)及心房颤动并右房扩大者(AF+RAD)左、右心房肌细胞Cx43mRNA和Cx43表达的变化、探讨Cx43mRNA和Cx43表达的变化及在房颤中的作用。方法：选择26例接受冠状动脉旁路移植术的冠心病患者和20例接受换瓣术的风心病患者为研究对象,46例患者术前均接受冠状动脉造影检查,根据心电图和心脏彩超资料分为四组：AF+LAD组26例,AF+RAD组20例,SR+LAD组8例,SR组12例,分别于手术中切取左、右心耳标本(左心房大小正常者一般只切取右心耳肌标本)。用RT-PCR法检测Cx43mRNA表达,用免疫印迹法(Western blot)检测Cx43蛋白的表达。结果：Cx43mRNA和Cx43表达量均值在四组患者的左、右心房肌标本中采用方差分析及两-两比较均无显著差异(P>0.05)。结论：1.不论是否发生左、右心房扩大和房颤,左、右心房肌细胞中Cx43mRNA表达无明显变化。2.不论是否发生左、右房扩大和房颤,左、右心房肌细胞中Cx43表达无明显变化,3.左、右心房扩大和房颤对Cx43mRNA和Cx43的表达均无明显影响,说明Cx43mRNA和Cx43表达的变化对房颤的发生影响较少。目的：制备光镜、电镜标本,应用光镜、电子显微镜观察心房肌细胞、细胞间质及缝隙连接的结构变化,探讨心房肌细胞、细胞间质及缝隙连接重构的变化在房颤发生中的作用。方法：选择26例接受冠状动脉旁路移植术的冠心病患者和11例接受换瓣术的风心病患者为研究对象,37例患者术前均接受冠状动脉造影检查,根据心电图和心脏彩超资料分为四组：AF+LAD组11例,AF+RAD组11例,SR+LAD组8例,SR组7例,分别于手术中切取左、右心耳标本(左心房大小正常者一般只切取右心耳肌标本)。观察心房肌细胞的形态和大小、细胞核的异形和大小、细胞间缝隙连接的变化、细胞间质纤维化的比例及脂肪浸润情况。结果：1.光镜下左、右心房扩大患者心房肌细胞肥大、细胞核肥大异形、细胞间质纤维化和脂肪浸润程度与左、右心房大小正常的患者比较有显著差异(P<0.01)。2.电镜下心肌细胞肌原纤维溶解、断裂；线粒体数量明显增多,且大小不一；细胞核异型明显；闰盘扭曲,盘旋重叠,模糊、不连续；心肌间质纤维增生明显与左、右心房大小正常的患者比较有显著差异(P<0.01)。结论：左、右心房扩大后,心房肌细胞、细胞间基质及细胞间缝隙连接发生了重构,心房结构重构是导致房颤的发生和维持的重要因素。更多还原

【Abstract】 Background:Atrial fibrillation is a kind of supraventricular arrhythmia which is a complete absence of coordinated atrial systole.commonly encountered arrhythmia. The mechanism of onset and maintenance of atrial fibrillation is not fully understood, though many recent studies have allowed improvements in the comprehension of the pathophysiology of the arrhythmia. For onset of atrial fibrillation, some researches on mechanism of onset dicovered the focal discharge could resulted in paroxysm AF in the vena cava system such as the vena coronaria, the superior and inferior vena cava and pulmonary vena. Besides, an increase in sympathetic tone, cardiac fat pad and vascular nerves in chambers heart is considered by many researchers as an important factor in initiating and maintaining atrial fibrillation, which first result in atrial premature beats, paroxysmal atrial tachycardia by diminish autorhythmic cell membrance potential, after-depolarization and triggers. For maintenance of atrial fibrillation, many recent studies have allowed atrial remodeling is the important reentry substrate of atrial fibrillation, which include atrial electroical remodeling and atrial anatomical remodeling. In current literatural, atrial fibrillation is generally subdivided into two forms:lone and pathological atrial fibrillation. Dilated left atrial has been considered to be one of the major factors linked to inducibility and persistence of pathological atrial fibrillation.Atrial enlargement may be related to both the multiple re-entrant circuits and electrophysiological characteristics of the atrial myocardium. In fact, all of myocardium is involved in any type of arrhythmia (including AF); arrhythmia is no fewer determined by passive current conduction than membrane channels. Accounting for AF is incomplete regardless of passive conduction between cells. It has been proved that gap junction is the only base structure responsible for intercellular current conduction. Gap junction is a specialized regions of the membranes of adjacent cells, containing arrays of densely packed intercellular channels that directly connect the cytoplasmic compartments of neighboring cells and permit ihtercellular passage of ions and small molecules. Gap junctions maintain cellular homeostasis by allowing communication between adjacent cells. In the heart, gap junctions provide the pathways for intercellular current flow, enabling coordinated action potential propagation. Gap-junctional channels are constructed from connexins (Cxs), a multigene family of conserved proteins termed connexins. In the mammalian heart, Single gap junction channel is composed of six connexin that abbreviate Cx by the molecular weight of the specific protein. It has been established that mammalian cardiac express Cx31.9, Cx37, Cx40, Cx43and Cx45. but different tissue of the heart express different amounts and combinations of these connexins, Cx43 was confirmed to be abundantly in all four chambers in human heart, Cx45 expression appears to be within the atrioventricular conduction system and Cx40 is present specifically in the atrium and in the specialized conducting system. Studies have shown that the changes of ionic concentration, ionic channel activities and phosphorylation have involved by the initiation of atrial fibrillation in a few seconds or minutes, and the expression changes of mRNA and proteins，including connexins, take place in several hours or days later. Structural changes are present in atrial fibrillation including atrium myocytes hypertrophia, apoptosis, inflammatory cell infiltrate and interstitial fibrosis.Cx40 gap junction channel was speculated to play critical roles in atria arrhythmia and AF. Although there are a few of researches about Cx40 and Cx43 on mechanisms of rheumatic heart disease AF, there are few reports about the changes of Cx40 and Cx43 expression, spatial distribution patern, and their morphology in human atrial myocardium from coronary heart disease. Our study composed of three parts aim to reveal the expression of Cx40, Cx40mRNA, Cx43, Cx43mRNA and the disorganization of connexin40 in the atrium myocardium of patients with coronary heart disease,, which to probe the mechanisms of atrial fibrillation in coronary heart disease. Objective:To explore the expression of Cx40mRNA、Cx40 in atrium myocardium of patients suffering from coronary heart disease with or without artial dilated or atrial fibrillation.Methods:26 patients with Coronary heart disease and 11 patients with rheumatic heart disease undergoing cardiac surgery for coronary artery bypass graft were involved in this study and were divided into four groups according to the atrial size and rhythm. All patients were examined by coronary arteriongraphy, echocardiogram and ECG before surgical operation.11 cases with AF and left atrial dilatation(AF+LAD),8 with sinus rhythm and left atrial dilatation (SR+LAD),7 sinus rhythm without atrial dilatation (SR) and 11 cases with AF and right artial dilatation(AF+RAD). Expression of Cx40mRNA was detected by RT-PCR. Expression of Cx40 was detected by immunoblotting assay(western blot).Results: 1. Significantly decreased were observed in the Cx40mRNA、Cx40 expression in atrium myocardium in all of the atrial dilatation groups (P <0.05).2. No obvious changes were observed in the Cx40mRNA、Cx40 expression in AF+LAD group and SR+LAD group(P> 0.05).3. Comparing with the Cx40mRNA、Cx40 expression in right atrium of the SR group, AF+LAD group and SR+LAD group, there was significantly decreased than that in the left atrium in AF+LAD group and SR+LAD group(P< 0.01).Conclusion:The decrease expression of Cx40mRNA、Cx40 is relation with left atrial dilatation, the decrease expression of Cx40mRNA、Cx40 and left atrial dilatation could be an important agents in the occurrence and maintenance of AF. Objective:To explore the effects of connexin 43mRNA and connexin 43 and the left atrium size in the AF by studying the expression of Connexin43mRNA and connexin 43 in Atrium of patients suffering from coronary heart disease with or without AF.Methods:26 patients with Coronary heart disease and 11 patients with rheumatic heart disease undergoing cardiac surgery for coronary artery bypass graft were involved in this study and were divided into four groups according to the atrial size and rhythm.11 cases with AF and left atrial dilatation (AF+LAD),8 with sinus rhythm and left atrial dilatation (SR+LAD),7 sinus rhythm without atrial dilatation (SR) and 11 cases with AF and right artial dilatation(AF+RAD). Expression of Cx43mRNA was detected by RT-PCR. Expression of Cx43 was detected by immunoblotting assay(western blot).Results:No obvious change was observed in the Cx43mRNA and Cx43 expression in dilated atrial myocytes in the four groups (P> 0.05). Conclusion:The expression of Cx43mRNA and Cx43 is not relation with left atrial dilatation and the expression of Cx43mRNA and Cx43 contribute less than the expression of Cx40 in the occurrence and maintenance of AF. Objective:The labelled samples were examined using a confocal microscope and light microscope to explore the anatomical structure of dilated atrium in the patients with coronary heart disease.Methods:26 patients with Coronary heart disease and 11 patients with rheumatic heart disease undergoing cardiac surgery for coronary artery bypass graft were involved in this study and were divided into four groups according to the atrial size and rhythm.11 cases with AF and left atrial dilatation (AF+LAD),8 with sinus rhythm and left atrial dilatation (SR+LAD),7 sinus rhythm without atrial dilatation (SR) and 11 cases with AF and right artial dilatation(AF+RAD). The spatial distribution pattern of Cx40 were detected through light microscope and confocal lasers canning microscopy assay.Results:1.The results revealed atrial anatomical remodeling have involved in the dilatation of atrial myocytes.2.Myolysis, glucogen aggregation, fibrosis, cytochondriom change and nuclear depolymerize have been observed by confocal microscope in dilated atrial myocytes.3. Adipose infiltration, hypertrophia, nuclear multiplication, nuclear atypia and interstitial fibrosis have been observed by light confocalConclusion:Atrial anatomical remodeling have involved in the dilatation of atrial myocytes which could be an important agent in the recurrence formation of AF.更多还原