第一部分  猪二尖瓣反流模型的特点

中文摘要

背景：二尖瓣反流模型根据模型来源分为：小动物二尖瓣反流模型、大动物二尖瓣反流模型、体外二尖瓣反流台架模型等；根据发病机制又分为原发性二尖瓣反流模型、继发性二尖瓣反流模型。这些模型各有特点，为临床研究提供差异化的支持。二尖瓣反流的大动物模型不仅能满足基础研究的需要，而且可用于外科手术器械、介入导管器械的研究开发和临床前评价，是心血管研究的重要工具。目前，随着疾病机制的研究深入和细化以及临床新的手术和介入器械的研发，二尖瓣反流的大动物模型有很大的需求量。均一性好和成功率高的标准化制造工艺，以及对模型的基础数据的系统研究，是未来研究对二尖瓣反流大动物模型的必然要求。猪与人的同源性高，心脏结构相似，而且具有成本低，方便饲养和管理等优点，是大动物实验选择最多的动物。因此猪的二尖瓣反流模型具有很广阔的应用前景。

目的：本研究旨在建立一种均一性好、成功率高、并发症少的猪二尖瓣重度反流模型。并对这些猪重度二尖瓣反流模型进行长期系统的研究，以确定其在心血管疾病中的潜在应用价值。

方法：17头猪被分为实验组(n=11)和对照组(n=6)。实验组采用自制腱索切割器经左侧胸部切口-左心耳-二尖瓣口入路，在食道超声探头经心外膜监测及左心房压力监测下，切断二尖瓣P2、P3区的腱索，使左心房压力升高10mmHg、反流束的面积占到左心房面积40%以上，建立重度二尖瓣反流模型，对照组进行与实验组相同的手术过程但不损伤二尖瓣腱索进行假手术操作。实验动物术后随访30个月，进行心电图、超声心动图、心内膜三维电生理标测、房颤易感性试验、心肌组织病理学等检测。

结果：二尖瓣反流手术死亡率为9%（1/11），模型成功率为82%（9/11），实验动物术后无严重并发症。术后实验组P波增宽，部分实验动物出现双峰改变。术后随访30个月，对二尖瓣反流组和对照组进行重复的超声心动图检测，左心房最大容积、左心房最小容积、左心房射血分数在两组间存在显著的组间差异、组内差异及交互作用；左心室收缩末容积和左心室舒张末容积在两组间无显著性的组间差异、组内差异及交互作用；左心室射血分数在两组间有显著性的组间和组内差异，但无交互作用。两组间左心房三维电生理标测图无明显差异。两组间左心房纤维化程度有显著差异性（胶原体积分数分别为18.1%±3.0%和1.6%±0.2%，P<0.05），而左心室纤维化程度无显著差异性（胶原体积分数分别为2.0%±0.5%和1.9%±0.3%，P=0.58）。两组间房颤易感性有显著性差异（P<0.05)。

结论：我们的方法可以简单而有效地制作猪重度二尖瓣反流模型，可用于二尖瓣反流的病理生理学研究，以及临床前手术器械的研发和评估。该模型也可用于房颤和心肌纤维化的研究，但不适用于心力衰竭的研究。

第二部分：猪二尖瓣反流所致心房纤维化对心房电生理及房颤易感性的影响

中文摘要

背景：房颤是一种室性上心律失常，其特点是快速、无序的心房电活动合并心房机械功能一定程度的下降，并引起心室不规则的跳动。房颤可以继发于多种心血管或非心血管疾病。心脏的结构重构和电重构被认为是房颤发生的基础。心房纤维化是心房结构重构的重要内容，并且在房颤过程中发挥了重要作用。然而，心房纤维化导致房颤发生和维持的机制尚不清楚。有些阵发性房颤患者心肌组织有大量的纤维化改变，而有些持续性房颤患者的心肌仅有轻微的纤维化改变。在以往关于房颤心房组织纤维化的研究中，心肌组织的标本常常是从心房单一的部位获取的，并不能反映心房纤维化的总体情况。

目的：检测心房组织不同区域的纤维化程度，研究纤维化和心房电传导、有效不应期的关系，探讨纤维化对房颤易感性的影响。

方法：将19头猪随机分为3组，对照组(n=6)进行假手术，实验组进行二尖瓣反流手术，并分别观察3个月(n=7)和6个月(n=6)。试验动物的右心房被分为右心耳、三尖瓣峡部、右心房前壁、右心房后壁、上腔静脉入口、下腔静脉入口6个区域，左心房被分为左心耳、房间隔、左心房后壁、左心房顶部、肺静脉口和二尖瓣峡部6个区域。通过房颤易感性试验评价实验动物的房颤易感性。通过三维电生理标测系统对双侧心房进行电生理标测，根据标测结果计算每个区域的心房电传导速度。根据三维心房模型引导，检测每个区域的有效不应期。通过组织病理学对每个区域进行纤维化评价。

结果：随着二尖瓣反流的持续，心房纤维化逐渐加重（对照组3.11%±0.08%，3个月组5.85%±0.42%，6个月组8.17%±0.23%，P<0.001），房颤易感性逐渐升高（对照组房颤易感性的阳性率0/6，3个月组2/7，6个月组5/6，P＜0.05），有效不应期逐渐延长（对照组220.1±1.1ms，3个月组244.4±1.4ms，6个月组289.0±8.9ms，P<0.001)，心房传导速度逐渐减慢（对照组为1.39±0.16m/s，3个月组为1.04±0.05m/s，6个月组为0.89±0.02m/s，P<0.001）。这些病理生理改变在心房不同部位是不均匀的。左心房纤维化程度高于右心房（3个月组：8.22%±0.83% vs 3.83%±0.25%，P<0.001；6个月组：11.76%±0.52% vs 5.09%±0.34%，P<0.001），左心房6个不同分区的纤维化值有显著性差异（3个月组和6个月均P＜0.001）；左心房平均传导速度低于右心房平均传导速度（3个月组：0.86±0.07 vs 1.20±0.32，P<0.001；6个月组：0.62±0.02 vs 1.13±0.03，P<0.001），左心房不同区域传导速度也具有显著性差异（3个月组和6个月均P＜0.001）；心房不同部位的有效不应期是不一致的，左心房平均有效不应期长于右心房（3个月组：251.4±2.9ms vs 238.6±3.1ms，P<0.001；6个月组：299.3±5.3ms vs 280.5±12.4ms，P<0.001）。房颤易感性与左心房纤维化的离散度有关。心房纤维化与心房传导速度呈显著负相关，而心房纤维化与心房有效不应期之间无明显相关性。

结论：随着二尖瓣反流时间的延长，心房组织纤维化程度增加，不同区域间的心房组织纤维化程度不一致。心房组织纤维化的离散度增加可能与房颤易感性升高有关。心房组织纤维化可能通过影响传导速度而不是有效的不应期来增加房颤易感性。

第三部分：慢性原发性二尖瓣反流不同阶段左心房功能的研究及其与手术适应症的关系

中文摘要

背景：慢性原发性二尖瓣反流是一种常见的瓣膜疾病，随着人口老龄化的进程，其发病率将会在未来几十年继续增加。手术是二尖瓣反流最有效的治疗方式，包括二尖瓣置换和二尖瓣修复手术。对于二尖瓣反流患者应该何时进行手术干预治疗是至关重要的，过早的手术会降低患者的生活质量，增加人工瓣膜和抗凝治疗的相关并发症，而过晚的手术会影响围手术期安全及远期预后。二尖瓣反流的手术适应症被持续修改，然而，目前仍然存在争议。在慢性原发性二尖瓣反流中，左心房是一个受累很重而又常常被忽略的器官。二尖瓣反流时左心房功能的评估，可能会为这些患者提供诊断价值。

目的：通过评估慢性原发性二尖瓣反流不同阶段的左心房功能参数，来探讨左心房参数在慢性原发性二尖瓣反流中的临床应用价值。

方法：选择133例窦性心律的慢性原发性二尖瓣反流患者，按二尖瓣反流程度分为轻-中度反流组与重度反流组，重度反流组再按照是否具有手术适应症分为早期-重度反流组与晚期-重度反流组，共三组。并与30例无心脏疾病的就诊人群作为对照组进行对比。使用超声心动图检查测量左心房最大容积（LAVmax）、左心房收缩前（P波前）容积（LAVp）、左心房最小容积（LAVmin），并由此推算出左心房扩张指数：LAEI=（LAVmax-LAVmin）/LAVmin×100%，左心房被动排空分数：LAPEF=（LAVmax-LAVp）/LAVmax×100%，左心房主动射血分数：LAAEF=（LAVp-LAVmin）/LAVp×100%，左心房总射血分数：TLAEF=（LAVmax-LAVp）/LAVmax×100%。并且通过超声心动图获取左心室舒张末容积、左心室收缩末容积、左心室射血分数。通过三尖瓣反流压差法或者肺动脉血流时间间期法检测是否肺动脉高压。通过询问判断患者是否有心功能不全的自觉症状。

结果：单因素分析显示，随着二尖瓣反流程度的增加，左心房容积（包括LAVmax、LAVp、LAVmin）逐渐增大，且各组间差异明显（P＜0.001）；左心房各项功能指标（包括LAEI、LAPEF、LAAEF、TLAEF）均逐渐降低，且各组间差异明显（P＜0.001）。LAVmax、LAVp、LAVmin、LAEI、LAAEF、TLAEF在早期-重度二尖瓣反流组和晚期-重度二尖瓣反流组之间有统计学差异，而LAPEF在两组间差异不明显（P=0.05）。logistic多因素分析显示，LAEI和LAVp是重度二尖瓣反流需要手术的独立预测因素。受试者工作特征曲线显示，LAEI的预测值最高，曲线下面积为0.91（95%可信区间，0.858~0.965），当LAEI≤97%预测二尖瓣手术适应症的敏感性和特异性分别为98%和67%。

结论：二尖瓣反流时左心房容积增大和功能降低与二尖瓣反流手术适应症之间具有相关性。通过LAEI预测二尖瓣手术适应症有良好的敏感性和特异性。超声心动图定量评估左心房功能可以作为判断二尖瓣反流手术时机的新指标。

part 1: the characteristics of a porcine mitral regurgitation model

abstract

background: mitral regurgitation model is divided into mitral regurgitation model in small animals, mitral regurgitation model in large animals, mitral regurgitation platform model in vitro according to the source, and into primary mitral regurgitation model and secondary mitral regurgitation model according to the pathogenesis. these models have their own characteristics to provide differential support for clinical research. the large animal model of mitral regurgitation can not only meet the needs of basic research, but also can be used in the research and development of surgical instruments, interventional catheter instruments and pre-clinical evaluation. it is an important tool for cardiovascular research. at present, with the research of the disease mechanism and the development of surgical and interventional instruments, the large animal model of mitral regurgitation has a great demand. the standardized manufacturing process with good consistency and high success rate, as well as the systematic research on the basic data of the model system, is the inevitable requirement for the animal model of mitral regurgitation in the future. in addition, pig has the advantages of high homology with human, similar heart structure, low cost, convenient feeding and management and so on. it is the most widely selected animal in large animal experiment. therefore, the model of mitral regurgitation in pigs has a wide application prospect.

aims: to establish a model of severe regurgitation of porcine mitral valve with good homogeneity, high success rate and less complications. to study these porcine models of severe mitral regurgitation in a long-term and systematic manner, and to discuss their potential applications in cardiovascular diseases.

methods: 17 pigs were divided into experimental group (n = 11) and control group (n = 6). in the experimental group, the chordae tendinosus in the p2, p3 region of the mitral valve were cut by a self-made chordae tendinous cutter through left thoracic incision-left atrial auricle-mitral orifice approach under the monitoring of epicardial ultrasound and left atrial pressure. during the operation, the left atrial pressure increased by 10 mm hg and the area of the regurgitation bundle accounted for more than 40% of the left atrium area, which resulted in the formation of a severe mitral regurgitation model. the control group underwent the same procedure as the experimental group but did not damage the chordae tendineae of the mitral valve for sham operation. the model animals were followed up for 30 months. electrocardiogram, echocardiography, endocardium three-dimensional electrophysiological mapping, atrial fibrillation susceptibility test and myocardial histopathology were measured.

results: the mortality of mitral regurgitation operation was 9% (1 / 11) and the success rate of the model was 82% (9 / 11). the ecg showed that the width of p wave increased in the experimental group, and some of the experimental animals showed double-pointed changes. during 30 months after operation, repeated echocardiography was performed in mitral regurgitation group and control group. there were significant inter-group differences, intra-group differences and interactions in left atrial maximum volume, left atrial minimum volume, left atrial ejection fraction between the two groups；no significant inter-group differences, intra-group differences and interactions in left ventricular end systolic volume and left ventricular end diastolic volume between the two groups；significant inter-group differences, intra-group differences but no interactions in left ventricular ejection fraction between the two groups. there was no significant difference in three-dimensional electrophysiological mapping of left atrium between the two groups. there was significant difference in the degree of left atrial fibrosis between the two groups (collagen volume fraction was 18.1% ±3.0% and 1.6% ±0.2%, p < 0.05), but no significant difference in the degree of left ventricular fibrosis (collagen volume fraction 2.0% ±0.5% and 1.9%, p=0.58). there was a significant difference in atrial fibrillation susceptibility between the two groups (p < 0.05).

conclusions: our method can be used to make a simple and effective porcine severe mitral regurgitation model, which can be used for the pathophysiological study of mitral regurgitation, as well as for the development and preclinical evaluation of instruments for mitral regurgitation. the model can also be used in the study of atrial fibrillation and myocardial fibrosis, but not not suitable in the study of heart failure.

part 2：effects of atrial fibrosis induced by mitral regurgitation on atrial electrophysiology and susceptibility to atrial fibrillation in pigs

abstract

background: atrial fibrillation is a kind of supraventricular arrhythmias characterized by rapid and disordered atrial electrical activity associated with a certain degree of decline in atrial mechanical function and causing irregular ventricular beats. atrial fibrillation can be secondary to a variety of cardiovascular or non-cardiovascular diseases. structural remodeling and electrical remodeling of the heart are considered as the basis for the occurrence of atrial fibrillation. atrial fibrosis is an important part of atrial structural remodeling and plays an important role in the process of atrial fibrillation. however, the mechanism of atrial fibrosis leading to atrial fibrillation is unclear. in some patients with paroxysmal atrial fibrillation, there is a large amount of fibrosis, while in some patients with persistent atrial fibrillation, there are only slight fibrosis changes. in previous studies, myocardial biopsies were often obtained from a single atrial site and did not reflect the overall condition of atrial fibrosis.

aims: to detect the degree of fibrosis in different areas of atrium, to study the relationship between fibrosis and atrial electrical conduction, effective refractory period, and to explore the effect of fibrosis on susceptibility to atrial fibrillation.

methods: 19 pigs were randomly divided into 3 groups: control group (n = 6) and experimental group (n = 6). mitral regurgitation was performed in the experimental group and observed for 3 months (n = 7) and 6 months (n = 6), respectively. the right atrium of the experimental animals was divided into 6 areas: right auricle, tricuspid isthmus, anterior wall of right atrium, posterior wall of right atrium, entrance of superior vena cava and entrance of inferior vena cava; and the left atrium is divided into 6 areas: left atrial ear, atrial septum, posterior wall of left atrium, top of left atrium, pulmonary vein orifice and mitral isthmus.

results: with the continuation of mitral regurgitation, atrial fibrosis aggravated (3.11% ±0.08% in the control group, 5.85% ±0.42% in the 3-month group and 8.17% ±0.23% in the 6-month group, p < 0.001)，atrial fibrillation susceptibility increased (0/6 in the control group，2/7 in the 3-month group, 5/6 in the 6-month group, p＜0.05)，the effective refractory period was prolonged (220.1 ±1.1 ms in control group, 244.4 ±1.4 ms in 3-month group, 289.0 ±8.9 ms in 6-month group, p<0.001), the conduction velocity decreased (1.39 ±0.16m/s in the control group, 1.04 ±0.05m/s in the 3-month group and 0.89 ±0.02m/s in the 6-month group, p < 0.001). these pathophysiological changes are uneven in different parts of the atrium. the degree of left atrial fibrosis is higher than that of right atrium (3.83%±0.25% vs 8.22%±0.83% in the 3-month group，p<0.001；5.09%±0.34% vs 11.76%±0.52% in the 6-month group，p<0.001); there was a significant difference in fibrosis in different regions of the left atrium (p＜0.001, in the 3-month and 6-momth group respectively). the mean conduction velocity of left atrium is lower than that of right atrium (0.86±0.07 vs 1.20±0.32 in the 3-month group，p<0.001；0.62±0.02 vs 1.13±0.03 in the 6-momth group，p<0.001), there was significant difference in conduction velocity in different regions of left atrium (p＜0.001, in the 3-month and 6-momth group respectively).the mean effective refractory period of the left atrium is longer than that of the right atrium (238.6±3.1ms vs 251.4±2.9ms in the 3-month group，p<0.001；299.3±5.3ms vs 280.5±12.4ms in the 6-momth group，p<0.001). susceptibility to atrial fibrillation is associated with dispersion of left atrial fibrosis. there was a significant negative correlation between atrial fibrosis and atrial conduction velocity, but no significant correlation between atrial fibrosis and atrial effective refractory period.

conclusion: with the prolongation of mitral regurgitation time, the degree of atrial fibrosis increased, and the degree of atrial fibrosis was different among different regions. the increased dispersion of atrial fibrosis may be associated with increased susceptibility to atrial fibrillation. atrial fibrosis may increase susceptibility to af by affecting conduction velocity rather than effective refractory periods.

part 3：changes of left atrial function in different stages of chronic primary mitral regurgitation and its relationship with surgical indications

abstract

background: chronic primary mitral regurgitation is the most common valvular disease and its incidence will continue to increase in the coming decades as the population ages. surgery is the most effective treatment for mitral regurgitation, including mitral valve replacement and mitral valve repair. optimum timing of surgical intervention is of paramount importance. premature surgery can reduce the patient's quality of life and increase complications associated with prosthetic valve and anticoagulation therapy, while too late operation will affect the perioperative safety and long-term prognosis. surgical indications for mitral regurgitation have been continuously modified, but remains clinically challenging. in chronic primary mitral regurgitation, the left atrium is a severely damaged and often neglected organ. assessment of left atrial function during mitral regurgitation may provide diagnostic value for these patients.

ive: to evaluate the clinical value of left atrial parameters in chronic primary mitral regurgitation by evaluating the parameters of left atrium in different stages of chronic primary mitral regurgitation.

methods: the study included 133 consecutive mitral regurgitation patients in sinus rhythm, classified into mild-moderate mitral regurgitation and severe mitral regurgitation groups according the degree of mitral regurgitation. then severe mitral regurgitation group was divided into early-severe group and late-severe group according to the indication of operation. the control group was 30 patients without cardiac disease. the left atrial maximum volume (lavmax), the left atrial volume before left atrial contraction (lavp), the left atrial minimum volume (lavmax) were measured by echocardiography. and then the left atrial dilatation index (ladi) is calculated as: ladi= (lavmax-lavmin) / lavmin × 100%, the left atrial passive emptying fraction (lapef) : lapef=（lavmax-lavp）/lavmax×100%，the left atrial active ejection fraction (laaef)：laaef=（lavp-lavmin）/lavp×100%，the total left atrial ejection fraction (tlaef)：tlaef=（lavmax-lavp）/lavmax×100%。left ventricular end-diastolic volume, left ventricular end-systolic volume and left ventricular ejection fraction were obtained by ultrasound. tricuspid regurgitation pressure difference or pulmonary artery flow time was used to detect pulmonary hypertension. patient's conscious symptoms of cardiac insufficiency was obtained by inquiry

results: univariate analysis showed that the left atrial volume (including lavmax, lavp, lavmin) increased with the aggravation of mitral regurgitation (p < 0.001). the left atrial functional parameters (including ladi, lapef, laaef, tlaef) decreased gradually with the aggravation of mitral regurgitation. there were significant differences among the three groups (p < 0.001). there was a significant difference in lavmax, lavp, lavmin, ladi, laaef, tlaef between early-severe and late-mitral mitral regurgitation group, but no significant difference in lapef between the two groups (p=0.05). logistic multivariate analysis showed that ladi and lavp were independent predictors of surgical indications for severe mitral regurgitation. receiver operating characteristic curve analyses showed that ladi has the highest predictive value, and the area under the curve is 0.91 (95% confidence interval, 0.858-0.965). when ladi was less than 97%, the sensitivity and specificity were 98% and 67%, respectively.

conclusion: during mitral regurgitation, left atrial volume enlargement and function decrease are correlated with the indications of mitral regurgitation surgery. ladi has good sensitivity and specificity in predicting the indication of mitral valve surgery. quantitative evaluation of left atrial function by echocardiography can be used as a new index to guide the timing of mitral regurgitation surgery.