【作者】 连亦田；

【导师】 程龙献；

【作者基本信息】 华中科技大学 ， 内科学， 2012， 博士

【摘要】 第一部分高血压合并房颤患者抗M2胆碱能受体抗体和抗肌球蛋白重链抗体的检测及其临床意义目的：自身抗体在心血管疾病中逐渐被发现,越来越多的证据表明自身免疫机制参与其中并发挥着重要作用,在心律失常中也是如此。本实验的目的是观察高血压合并房颤患者自身抗体抗M2胆碱能受体抗体(AAM2R)和抗肌球蛋白重链抗体(AAMHC)的变化以及二者与房颤的关系。方法：研究选取高血压患者共295例,分为高血压合并房颤组(145例)和高血压非房颤组(150例)。采集血样,通过ELISA方法检测血清中四种自身抗体(AAM2R/AAMHC/AAβ1R/AAANT)的阳性率,并进一步通过单变量和多变量logistic回归分析自身抗体与房颤的关系。研究还选取了健康体检者80例作为正常对照组,与上述两组高血压患者比较自身抗体阳性率的差异。结果：与高血压非房颤组患者相比,高血压合并房颤组患者自身抗体AAM2R和AAMHC的阳性率显著升高(AAM2R:44.8%vs13.3%:AAMHC:27.6%vs14.7%:P<0.01),并且自身体阳性组患者房颤的发病率较抗体阴性组也显著增加(AAM2R:76.5%vs38.1％,P<0.001;AAMHC:64.5%vs45.1%,P<0.01)；多变量分析显示自身抗体是高血压患者发生房颤的危险因素和强预测因子(AAM2R:OR=5.251; AAMHC:OR=2.696)。正常对照组抗体阳性率显著低于上述两组高血压患者(P<0.05)。结论：自身抗体AAM2R和AAMHC与高血压患者房颤的发生密切相关,是其重要危险因素和强预测因子,这可能提示自身免疫参与并促进了房颤的发生与维持,在其中发挥了重要作用。第二部分抗M2胆碱能受体抗体和抗肌球蛋白重链抗体的提取、纯化和鉴定目的：在高血压合并房颤患者体内自身抗体AAM2R和AAMHC有较高检出率,本部分实验主要目的是验证两种自身抗体与相应抗原结合的特异性。方法：利用心外科开胸手术患者(窦性心律)右心耳组织标本,提取组织蛋白和酶解分离单个心房肌细胞。采用盐析法和亲和层析法提纯血清中的抗体,利用免疫印迹和细胞免疫荧光技术验证自身抗体与相应抗原的特异性结合。结果：自身抗体AAM2R能够与人心房组织蛋白M2受体和表达于CHO细胞的重组人M2受体特异性结合,免疫荧光共聚焦显微镜下可见AAM2R能与人心房肌细胞膜表面相结合,抗体阴性患者血清提取物和用抗原中和后的抗体未能结合；自身抗体AAMHC可与猪心肌肌球蛋白重链和人心房肌球蛋白重链特异性结合,并且免疫荧光共聚焦显微镜下可见AAMHC能与经破膜处理的人心房肌细胞结合,抗体阴性患者血清提取物和用抗原中和后的抗体未能结合；自身抗体AAMHC与人β1受体可发生交叉反应。结论：高血压合并房颤患者血清中存在特异性的自身抗体AAM2R和AAMHC,能够分别与人心房肌M2受体和肌球蛋白重链结合,且自身抗体AAMHC与β1受体具有交叉反应。第三部分抗M2胆碱能受体抗体和抗肌球蛋白重链抗体对人心房肌细胞复极电流及动作电位的影响目的：自身抗体AAM2R和AAMHC与高血压患者房颤发生密切相关,是其重要危险因素和强预测因子。本部分实验探讨两种自身抗体对人心房肌细胞复极化电流和动作电位的影响,以阐明这两种自身抗体致心律失常的电生理作用机制。方法：采集窦性心律心脏外科手术患者右心耳组织,酶解分离单个心房肌细胞；利用全细胞膜片钳技术和穿孔膜片钳技术分别记录两种自身抗体对心房肌细胞各种复极化电流(Ikach、ICa-L、Itol、Ikur)的影响；利用全细胞穿孔膜片钳电流钳模式观察两种自身抗体对心房肌细胞动作电位的影响。结果：与自身抗体阴性血清提取物相比,自身抗体AAM2R产生了受体激动样作用,可使心房肌细胞Ikach通道开放,使ICa-L电流减少,并缩短动作电位时程,但对Itol、Ikur电流无明显影响,用相应M2受体抗原肽段中和抗体后,此作用消失；自身抗体AAMHC对ICa-L、Itol、Ikur电流及心房肌细胞动作电位均无影响。结论：自身抗体AAM2R改变了心房肌细胞离子通道的电生理特性,使之发生重构,从而有利于房颤的发生与维持,而自身抗体AAMHC未能产生这种作用,可能是通过其他机制促发房颤。因此,研究结果表明自身免疫机制与房颤密切相关,在其中发挥着重要作用,可能为将来房颤的治疗提供了新的靶点。更多还原

【Abstract】 Part I The detection and clinical significance of anti-M2R autoantibody and anti-cardiac mysosin heavy chain autoantibody in hypertensive patients with atrial fibrillationObjective:To investigate the change of anti-M2R autoantibody and anti-cardiac mysosin heavy chain autoantibody in hypertensive patients with atrial fibrillation and elucidate the relationship between both autoantibodies and atrial fibrillation.Methods:The selected295hypertensive patients were divided into two groups:group one consisting of145cases with atrial fibrillation, and group two consisting of150cases without atrial fibrillation used as control group. Peripheral blood samples were collected, and then tested for the detection of four autoantibodies (AAM2R, AAMHC, AAβ1R and AAANT) by using enzyme-linked immunoabsorption assays (ELISA). At last, the relationships between autoantibodies and atrial fibrillation were analyzed through univariate and multivariate logistic regression analysis. We also selected80subjects for health examination as normal control group, in order to observe the differences of autoantibodies detection with the above groups.Results:Compared with group two (control group), the positive rate of both autoantibodies (AAM2R and AAMHC) significantly increased in hypertensive patients with atrial fibrillation (AAM2R:44.8%vs13.3%; AAMHC:27.6%vs14.7%; P<0.01). The incidence of atrial fibrillation in autoantibody-positive hypertension patients remarkable increased in contrast with autoantibody-negative patients(AAM2R:76.5%vs38.1%, P<0.001; AAMHC:64.5%vs45.1%, P<0.01). Autoantibodies both AAM2R and AAMHC were significant risk factors and strong predictors (AAM2R:OR=5.251; AAMHC:OR=2.696). The positive rate of normal control group was significantly lower than that of the two groups of hypertensive patients.Conclusion:The present study indicated that autoantibodies both AAM2R and AAMHC were closely associated with atrial fibrillation in hypertensive patients, suggesting that autoimmune mechanism might play a crucial role in the initiation and development of atrial fibrillation. Part Ⅱ The extraction, purification and identification of anti-M2receptor and anti-cardiac mysosin heavy chain autoantibodies from hypertensive patients with atrial fibrillationObjective:There were highly detection of autoantibodies both AAM2R and AAMHC in the sera from hypertensive patients with atrial fibrillation. The goal of this study was to determine the binding specificity of both autoantibodies with corresponding antigen respectively. Methods:The right atrial appendages from patients underwent cardiac open-chest surgery were extracted for whole proteins and enzymatically isolated for single atrial myocytes. By using salt precipitation in (NH4)2SO4and affinity chromatography, the autoantibodies were purified from the sera of hypertensive patients.The binding specificities of both autoantibodies with corresponding antigen were confirmed through immunoblotting and cellular immunofluorescence techniquesResults:AAM2R specially reacted with M2receptors of human atrial tissue and recombinant human M2receptor expressed in CHO cells. By using laser confocal immunofluorescence, AAM2R specially reacted with human atrial myocytes. The negative sera and autoantibody absorbed by corresponding antigen failed to do so. There were similar results of AAMHC by immunoblotting and immunofluorescence. Furthermore, AAMHC could cross-reacted with human β1receptor.Conclusion:There were special autoantibodies of both AAM2R and AAMHC in the sera from hypertensive patients with atrial fibrillation. They could specially be bound with human atrial M2receptor and myosin heavy chain respectively. AAMHC could cross-reacted with human β1receptor. Part III The effects of anti-M2receptor and anti-cardiac mysosin heavy chain autoantibodies on repolarization currents and action potential of human atrial myocytesObjective:Autoantibodies both AAM2R and AAMHC were closely associated with atrial fibrillation in hypertensive patients, which were important risk factors and strong predictors of atrial fibrillation. the aim of the present study is to explore the influence of autoantibodies on the repolarization current and action potential of huaman atrial myocytes, through which the electrophysiological mechanism of arrhythmogenesis of autoantibodies could be elucidated.Methods:Right atrial appendages were obtained from cardiac surgical patients with sinus rhythm, which were enzymatically isolated for single atrial myocytes. The effects of autoantibodies on a variety of repolarization currents of human atrial myocytes were recorded by using whole-cell and perforated patch clamp techniques. The effects of autoantibodies on action potential of atrial myocytes were observed by current-clamp mode with perforated patch clamp technique.Results:In contrast with the autoantibody-negative sera, AAM2R produced muscarinic agonist-like actions, such as the open of Ikach channel, the reduction of ICa-L current, the shortening of action potential duration, which were abolished upon autoantibody absorption using corresponding antigenic peptide. However, AAM2R failed to change Itol and Ikur currents. The effects of AAMHC on the above currents and action potential of atrial myocyte were not observed.Conclusion:the alteration of electrophysiological properties of human atrial myocyte ion channels by AAM2R could occurred, which was contributed to atrial electrical remodeling, leading to the initiation and perpetuation of atrial fibrillation. However, AAMHC failed to do so and perhaps influence atrial fibrillation via other mechanism such as inflammation. Our study suggested autoimmune might took part in the initiation and development of atrial fibrillation.更多还原