【作者】 肖培林；

【导师】 殷跃辉；

【作者基本信息】 重庆医科大学 ， 内科学， 2011， 博士

【摘要】 心房颤动（atrial fibrillation, AF）是临床上最常见的心律失常之一,是心血管事件的独立危险因素。AF常导致心功能下降和动脉血栓栓塞,具有发病率高、致残率高、致死率高、花费高的特点,严重影响患者生活质量。随着人口老龄化,AF的危害将越发严重,必将带来巨大的社会和经济负担。研究表明,RAS激活在AF的发生和维持中扮演着重要角色。动物实验和临床研究均显示RAS抑制剂可降低AF的发生率。ACEI可降低心房压,减少房性早搏,减轻心房纤维化,降低AF复律后复发。AF是甲亢最常见的并发症之一。10%-15%的甲状腺功能亢进患者合并AF,而不明原因的AF患者大约有13%伴有甲状腺素生化指标的异常。有的患者在甲状腺功能正常后转为窦性心律,而部分患者在甲状腺功能完全正常后三个月内AF仍然存在,表明有引起AF的基质存在。因此,弄清高甲状腺素引起AF基质的机制对于预防和治疗高甲状腺素继发的AF具有重要的意义。研究提示,甲状腺素可通过多种途径引起RAS激活。目前,对高甲状腺素下易患房颤的机制研究主要集中在甲状腺素的直接毒性作用上,对于甲状腺素是否通过RAS间接引起AF鲜有报道;关于RAS抑制剂是否可以通过抑制RAS激活改良高甲状腺素诱导的AF基质尚无报道。基于上述理论及存在的问题,本研究拟通过腹腔注射左旋甲状腺素建立高甲状腺素兔模型,然后给予RAS抑制剂贝那普利和厄贝沙坦,检测高甲状腺素模型兔心房电生理特性变化,了解RAS抑制剂是否可以改善高甲状腺素诱导的异常电生理特性,降低AF发生;与此同时,通过检测心房结构重构,离子通道重构和缝隙连接重构等变化明确RAS抑制剂对高甲状腺素诱导的异常电生理特性影响的潜在机制;在此基础上,我们还将通过差异表达蛋白组学技术,全面的探讨高甲状腺素致AF的机制及RAS在其中扮演的角色。共包括以下三个部分。第一部分RAS抑制剂对高甲状腺素诱导的心房异常电生理特性的影响目的:探讨RAS抑制剂对高甲状腺素诱导的异常电生理特性和房颤诱发率的影响。方法:将40只新西兰大白兔随机分为四组（n=10）:正常对照组,高甲状腺素组,贝那普利组和厄贝沙坦组。正常对照组和高甲状腺素组分别腹腔注射等量生理盐水和左旋甲状腺素（50μg/kg/d）。贝那普利组和厄贝沙坦组在腹腔注射左旋甲状腺素（50μg/kg/d）的同时灌服贝那普利（1mg/kg/d）或厄贝沙坦（10mg/kg/d）。4w后行心内电生理检测,评价AERP、AERP的频率适应性和房颤诱发率。结果:在高甲状腺组房颤诱发率为75%,显著高于正常对照组10%。给予贝那普利或厄贝沙坦显著降低房颤诱发率,分别为37%和44%。高甲状腺素组AERP200较正常对照组明显缩短,分别为97.10±4.75ms和75.13±5.41ms （ P<0.01）。但在高甲状腺素组、贝那普利组和厄贝沙坦组三组间AERP200无明显差异,分别为75.13±5.41ms、76.63±4.44ms和79±4.95ms。AERP150和AERP130在各组间的趋势与AERP200相同。正常对照组的AERP的频率适应性正常,当基础周长从200ms缩短为130ms时,AERP200较AERP130缩短了12.70±2.95ms。而高甲状腺素组,当基础周长从200ms缩短为130ms时,AERP200较AERP130缩短了6.25±2.55ms,明显小于正常对照组,且差异有统计学意义（P<0.01）。给予贝那普利或厄贝沙坦明显抑制甲状腺素引起这种变化。结论:贝那普利和厄贝沙坦可以部分改良高甲状腺素诱导的异常电生理特性,降低房颤诱发率。第二部分RAS抑制剂对高甲状腺素诱导的心房重构的影响第一节RAS抑制剂对高甲状腺素诱导的心房结构重构的影响目的:探讨RAS抑制剂对高甲状腺素诱导的心房结构重构的影响。方法:将40只新西兰大白兔随机分为4组,分组及处理方式同第一部分。4w后,取左心房组织,10%的多聚甲醛中固定24h,常规脱水,透明,石蜡包埋,切片（4μm）,行HE染色和Masson染色,普通光镜观察各组病理结构变化,计算心房肌胶原容积分数,评价各组心房肌纤维化程度。结果:HE染色见正常对照组肌节排列和细胞间隙均正常。甲状腺素组心肌纤维增粗、紊乱和断裂,细胞间隙增大,肌纤维分散、坏死,明显的纤维组织增生,脂肪变性,脂肪组织增多,少许炎性细胞浸润。贝那普利组和厄贝沙坦组细胞形态排列与高甲状腺素组相似,但上述异常病理变化较甲状腺素组明显减轻。Masson染色见高甲状腺素组蓝色胶原纤维较正常对照组明显增多,心房肌胶原容积分数分别为7.3±1.3%和17.1±2.2%,P<0.01;而贝那普利和厄贝沙坦明显抑制高甲状腺素诱导的胶原纤维增多,心房肌胶原容积分数在贝那普利组和厄贝沙坦组分别为12.3±1.8%和11.7±1.2%,与甲状腺素组比有统计学意义,P<0.01。结论:贝那普利和厄贝沙坦可改善高甲状腺素诱导的心房结构重构。第二节RAS抑制剂对高甲状腺素诱导的心房离子通道重构的影响目的:探讨RAS抑制剂对高甲状腺素诱导的心房离子通道重构的影响。方法:将40只新西兰大白兔随机分为4组,分组及处理方式同第一部分。4w后,取左心房组织,常规方法提取RNA和蛋白质,通过Real time PCR和Western blot检测L型Ca²⁺通道电流和Ito电流相关亚基基因和蛋白变化。结果:高甲状腺素组Cav1.2和Cav1.3mRNA表达较正常对照组明显降低,贝那普利和厄贝沙坦均可明显抑制甲状腺素引起的这种改变。Western blot结果显示,Cav1.2蛋白在各组间的变化与mRNA的变化同步。与正常对照组比较,高甲状腺素组kv1.4 mRNA表达明显增高,而kv4.2和kv4.3 mRNA表达在两组间差异无显著性。贝那普利组和厄贝沙坦组kv1.4、kv4.2和kv4.3 mRNA表达较高甲状腺组明显增高。Western blot结果显示,kv4.2蛋白在各组间的表达与mRNA表达趋势一致。结论:贝那普利和厄贝沙坦可抑制高甲状腺素诱导的L型Ca²⁺通道电流相关亚基的减少,而对Ito电流相关亚基的变化无明显抑制作用。第三节RAS抑制剂对高甲状腺素诱导的缝隙连接重构的影响目的:探讨RAS抑制剂对高甲状腺素诱导的缝隙连接重构的影响。方法:将40只新西兰大白兔随机分为4组,分组及处理方式同前。4w后,取左心房组织,通过Real time PCR和Western blot检测Cx43和Cx40基因和蛋白在各组中的变化,通过免疫荧光技术检测Cx43和Cx40在各组心房肌细胞中的分布特点。结果:Cx43 mRNA表达在高甲状腺素组较正常对照组明显增加,而Cx40 mRNA在两组间无明显差异,贝那普利和厄贝沙坦增加Cx43和Cx40 mRNA的表达。在蛋白水平,高甲状腺素组Cx40蛋白表达较正常对照组明显降低,贝那普利和厄贝沙坦明显增加Cx40蛋白的表达,Cx43蛋白表达与mRNA趋势一致。免疫荧光发现,甲状腺素组Cx40和Cx43呈异质性分布,排列紊乱,端端连接比例较正常对照组明显减少。贝那普利和厄贝沙坦能明显减轻缝隙连接的异质性分布和排列紊乱,增加端端连接比例。结论:贝那普利和厄贝沙坦可明显改善高甲状腺素诱导的Cx43和Cx40密度和分布异常。第三部分高甲状腺素诱导心房颤动基质的蛋白组学研究目的:采用蛋白组学技术全面探讨甲亢易患AF的机制,了解RAS激活在其中的作用。方法:将30只新西兰大白兔随机分为三组（n=10）,正常对照组、高甲状腺素组和厄贝沙坦组,处理方法同前。4w后,取右心房组织,提取总蛋白,样品制备,行2-DE,银染,照相。所有2-DE完成后通过PDQest软件分析,找出差异蛋白点。然后,通过MALDI-TOF-MS-MS鉴定差异蛋白点。最后,采用生物信息学手段分析相关蛋白的结构、功能以及与高甲状腺素致房颤的相关性。结果:通过PDQuest软件分析发现2-DE结果重复性较好,匹配率为88.6%。正常对照组、高甲状腺素组和厄贝沙坦组的蛋白点数分别为:1314±58、1364±69和1287±47,各点在图中分布均匀。应用PDQuest软件对各组的标准凝胶图像之间进行配比分析,共发现25个差异蛋白点。与正常对照组相比,高甲状腺素组有11个蛋白增加,10个蛋白减少,4个蛋白消失。厄贝沙坦组较高甲状腺素组有7个蛋白增加, 13个蛋白降低,1个新增。通过MALDI-TOF-MS-MS鉴定出21个蛋白点,19种蛋白,其中13种蛋白可能参与了高甲状腺素致AF基质的形成。结论:多种蛋白可能参与了高甲状腺素致AF基质的形成;RAS激活参与了部分AF基质的形成。更多还原

【Abstract】 Atrial fibrillation （AF） is the most common arrhythmia in clinical practice representing an independent risk factor for cardiovascular events. AF often results in cardiac insufficiency and artery embolism with characters of high morbidity, high disability, high mortality and high cost, which seriously affect the quality of the patients’life. With the population aging, the harm of AF will continue to grow and bring great social and economic burden.RAS plays an important role in the promotion and maintainance of AF. Experimental and clinical study suggested RAS inhibitor could reduce the incidence of AF. ACEI could reduce the atrial pressure, the atrial premature beat, the atrial fibrosis and the recurrence after cardioversion.AF is the most common cardiac complication of hyperthyroidism. AF occurs in 10%-15% of patients with hyperthyroidism. 13% of AF patients without definite cause suffer from disorder of thyroxine related biochemistry index. Some patients can convert to sinus rhythm after thyroid function resume normal. But for some patients, AF still exists even three months later after thyroid function resume normal, which suggests there exists the AF substrate. Therefore, make clear the mechanism has the vital significance in the prevention and treatment of AF underlying hyperthyroidStudies suggested that thyroxine could result in RAS activation by many pathway. However, present studies were mainly concentrated on direct toxic effects of thyroxine. whether RAS activation indirectly involved in the substrate AF was seldom reported. There were no study on whether RAS inhibitors could improve the substrate of AF underlying hyperthyroid.Based on the above mentioned theoretical basis and problems, this study was designed as follows: firstly, hyperthyroid rabbit model was established by intraperitoneal injection levorotatory thyroxine（L-Thy）, and given RAS inhibitors: benazepril or irbesartan; Then atrial electrophysiological properties were detected to identify whether RAS inhibitors could improve the abnormal electrophysiological properties underlying hyperthyroid, thus reducing the occurrence of AF; Meanwhile, the atrial structural remodeling, ion channels remodeling and gap junctions remodeling were evaluated to explore the potential mechanism of RAS activation influence on hyperthyroid rabbit; Based on above, proteomics study was performed to comprehensive analysis of the mechanism of hyperthyroid susceptibility to AF and the role of RAS.Overall,The study was composed of the following three parts. PARTⅠTHE EFFECTS OF RAS INHIBITORS ON ABNORMAL ELECTROPHY- SIOLOGICAL PROPERTIES UNDERLYING HYPERTHYROIDObjective: To explore the effects of RAS inhibitors on abnormal electrophysiological properties underlying hyperthyroid and the occurrence of induced AF.Methods: 40 New Zealand white rabbit were randomly divided into four groups （n = 10） : sham group, thyroxine group, benazepril group and irbesartan group. The rabbits in sham group and thyroxine group only received peritoneal injection saline or peritoneal injection L-Thy 50ug/kg/day for four weeks respectively. The rabbits in benazepril and irbesartan group received peritoneal injection L-Thyroxine （50 ug/kg/day） and benazepril （1 mg/kg/day） or irbesartan （10 mg/kg/day） orally for four weeks. 4w later, intracardiac electrophysiologic study was performed to evaluate AERP, physiologic rate adaptation of AERP and the occurrence of induced AF.Results: the occurrence of induced AF in thyroxine group was significantly higher than sham group （75% vs 10%）, and administered benazepril or irbesartan significantly reduced it （37% and 44%,respectively）. Compared with the sham group, the AERP200 in thyroxine group was significantly shortened （97.10±4.75ms vs 75.13±5.41ms, P<0.01）. However, no significant difference was found in AERP200 among thyroxine group, benazepril group and irbesartan group （75.13±5.41ms vs 76.63±4.44ms, 79±4.95ms, P=0.28）. AERP150 and AERP130 in each group had the same trend with AERP200. The physiologic rate adaptation of the AERP was normal. The AERP130 shortened for 12.70±2.95ms in contrast to AERP200. But the difference in thyroxine group was significantly shorter compared with sham group（6.25±2.55ms vs 12.70±2.95ms）. Administered benazepril or irbesartan significantly inhibited the shorten.Conclusion: Benazepril or irbesartan could partly improve abnormal electrophysiological properties underlying hyperthyroid and thus reduce the occurrence of AF. PARTⅡTHE EFFECTS OF RAS INHIBITORS ON ATRIAL REMODELING UNDERLYING HYPERTHYROIDSECTIONⅠ: THE EFFECTS OF RAS INHIBITORS ON ATRIAL STRUCTURAL REMODELING UNDERLYING HYPERTHYROIDObjective: To investigate the effects of RAS inhibitors on atrial structural remodeling underlying hyperthyroid.Methods: 40 New Zealand white rabbits were randomly divided into four groups. The group and treatment strategy were the same to part 1. 4w later, left atrial free wall was carefully dissected from all tissue preparations, and immersed in 10% phosphate-buffered formalin for 24h. After dehydration, each section was cut into 4μm-thick slices. Deparaffinized sections were stained with HE and Masson. Observed the pathological structure changes by microscopy and calculated collagen volume fraction in each groups to evaluate atrial fibrosis.Results: Atrial myocyte from sham rabbits showed a normal composition of sarcomeres distributed throughout the cell, and the intracellular space also appeared normal. Hyperplasia, disturbance, fragment of myocyte fiber, widen intracellular space, inflammation cell filtration and obvious fibrous connective tissue accrementition were observed in thyroxine group. However, these changes were significantly attenuated by benazepril or irbesartan. Masson dyeing showed blue collagen fiber in thyroxine group significantly increased compared with sham group （collagen volume fraction: 17.1±2.2% vs 7.3±1.3%, P<0.01, for sham group and thyroxine group respectively）. Benazepril or irbesartan group could significantly restrain the increase （collagen volume fraction: 12.3±1.8% , 11.7±1.2% vs 17.1±2.2%,P<0.01, for benazepril group, irbesartan group and thyroxine group respectively）.Conclusion: Benazepril or irbesartan could improve atrial structural remodeling underlying hyperthyroid.SECTIONⅡ: THE EFFECTS OF RAS INHIBITORS ON ATRIAL ION CHANNEL REMODELING UNDERLYING HYPERTHYROIDObjective: To investigate the effects of RAS inhibitors on atrial ion channel remodeling underlying hyperthyroid.Methods: 40 New Zealand white rabbits were randomly divided into four groups. The group and treatment strategy were the same to part 1. 4w later, left atrial free wall was carefully dissected from all tissue preparations. Extracted RNA and protein with routine methods. The real-time PCR and western blot was performed to detect the expression of L-type Ca²⁺ channel and Ito current related subunit.Results: The mRNA expression of Cav1.2 and Cav1.3 in thyroxine group significantly reduced compared with sham group. Benazepril and irbesartan could significantly inhibite these reductions. The result of western blot demostated the protein expression of Cav1.2 parallelled with the mRNA expression. The mRNA expression of kv1.4 in thyroxine group was higher than in sham group. However, no significant difference were seen for the mRNA expression of kv4.2 and kv4.3. Benazepril and irbesartan significantly increased these mRNA expression. The result of western blot showed that the protein expression of kv4.2 was consistent with its mRNA expression.Conclusion: Benazepril and irbesartan could inhibit the reduction of L-type Ca²⁺ channels related subunit, but couldn’t inhibit the changes of Ito related subunit underlying hyperthyroid. SECTIONⅢ: THE EFFECTS OF RAS INHIBITORS ON ATRIAL GAP JUNCTION REMODELING UNDERLYING HYPERTHYROIDObjective: To investigate the effects of RAS inhibitors on atrial gap junction remodeling underlying hyperthyroid.Methods: 40 New Zealand white rabbits were randomly divided into four groups. The group and treatment strategy were the same to part 1. Four weeks later, left atrial free wall was carefully dissected from all tissue preparations. The real-time PCR and western blot was performed to detect the expression of Cx43 and Cx40. The fluorescent immunohistochemistry was performed to detect the distribution of Cx43 and Cx40.Results: The mRNA expression of Cx43 in thyroxine group significantly increased compared with sham group, but no obvious difference was shown for Cx40. Benazepril and irbesartan increased the mRNA expression of Cx43 and Cx40. In the protein level, the expression of Cx40 in thyroxine group significantly lower than sham group. Benazepril and irbesartan significantly increased the protein expression of Cx40. The protein expression of Cx43 was consistent with the mRNA expression. Fluorescent immunohistochemistry found that thyroxine group Cx43 and Cx40 displayed with a heterogeneous distribution, arranging disorderly and obvious reduction of polar connection. Benazepril and irbesartan significantly extenuated the gap junctions heterogeneity distribution and arranging disorderly, and increased the proportion of polar connection.Conclusion: Benazepril or irbesartan could improve the abnormal expression and distribution of Cx43and Cx40.PARTⅢPROTEOMICS STUDY ON THE SUBSTRATE OF THE ATRIAL FIBRILLATION UNDERLYING HYPERTHYROIDObjective: To comprehensive explore the mechanism of hyperthyroid susceptibility to AF and the role of RAS activation in it.Methods: 30 New Zealand white rabbits were randomly divided into three groups （n = 10） : sham group, thyroxine group and irbesartan group The treatment strategy were the same to part 1. Four weeks later, the right atrial free wall was carefully dissected from all tissue preparations;Prepared sample with routine methods; Performed 2-DE and silver stain; Took picture; Detected differential proteins points with PDQest soft; Identificated proteins with MALDI-TOF-MS-MS; Then, analyzed the construction and function of these protein, and evaluated the association between these protein and hyperthyroid susceptibility to atrial fibrillation.Results:The results of 2-DE had high repeatability and the matching rate was 88.6%. The protein spot number was 1314±58, 1364±69 and 1287±47 in sham group, thyroxine group and irbesartan group respectively. Matched and analyzed with PDQuest soft and found 25 differential protein spots. 8 protein spots increased, 10 protein spots decreased and 4 protein spots disappeared were seen in thyroxine group compared with sham group. There were 7 protein spots increased, 13 protein spots decreased and 1 new protein spots disappeared in irbesartan group compared with thyroxine group. 21 protein spots （19 protein） was successfully identified , and we believed there were 13 protein being associated with the substate of AF underlying hyperthyroid.Conclusion:Many proteins were involved in the substrate of AF underlying hyperthyroid. RAS activation was associated with part of these substrates.更多还原