【作者】 李欣；

【导师】 徐波；

【作者基本信息】 华东师范大学 ， 运动人体科学， 2011， 博士

【摘要】 中等强度耐力训练诱导的运动性心肌肥大虽然体现了人体对系统训练的生理适应,但仍存在一些病理性的改变,具有潜在的疾病和猝死的风险。那么中等强度耐力训练诱导的运动性心肌肥大其性质究竟如何；此时心脏生理性肥大的典型信号通路(PI3K/Akt/mTOR信号通路)、心脏病理性肥大的典型信号通路(以心脏血管紧张素Ⅱ及其1型受体为代表的Gq信号通路)以及其它相关信号通路中各信号分子的表达究竟是如何变化的,各相关信号通路在此时致肥大信号网络中的具体作用如何,目前仍未见系统、明确的研究报道。目的：通过8周中等强度耐力训练,根据大鼠心脏损伤标志物(心肌肌钙蛋白T、心肌肌钙蛋白Ⅰ和脑钠肽)的表达变化,结合组织学表型,判断中等强度耐力训练诱导的耐力型运动心脏性质；通过心脏局部血管紧张素Ⅱ及其1型受体mRNA表达的变化情况,结合我们前期的研究成果,明确这条心脏病理性肥大的代表性信号通路此时的变化机制,阐明心脏局部血管紧张素Ⅱ及其1型受体的变化对此种心肌肥大的具体作用；通过心脏PI3K/Akt/mTOR信号通路中各信号分子表达的变化情况,明确这条心脏生理性肥大的代表性信号通路此时的具体变化情况,从整体上阐明该信号通路的变化机制。进而明确中等强度耐力训练诱导的心肌肥大时肥大信号网络中各信号通路分子所扮演的角色,为运动心脏的信号通路和内分泌研究提供更新的形态和生理生化理论依据。对科学制订运动训练计划,加强运动员心脏的保护和监制,制订心血管疾病的运动处方,提高体育人口心脏健康,积极防治心血管疾病也具有一定的参考价值。方法：(1)60只雄性SD大鼠(3月龄)随机分为对照组(C组,n=30)和中等强度耐力训练组(E组,n=30),每组包括形态学检测亚组(C1/E1组,n=10)、ELISA检测亚组(C2/E2组,n=10)和实时荧光定量PCR检测亚组(C3/E3组,n=10)。E组大鼠通过8周中等强度的跑台耐力训练,建立大鼠耐力型运动心脏模型。训练计划按照Fenning的训练方案,结合Wisloff对最大摄氧量的控制方法,相对运动强度大约维持在60%-65%VO2max。(2)通过实时荧光定量PCR技术检测C3和E3组大鼠心脏PI3K/Akt/mTOR信号通路中各信号分子PI3K、PDK1、Akt、mTOR、p70S6K、eIF4E和AMPK以及Gq信号通路(以局部血管紧张素Ⅱ及其1型受体为代表)的mRNA表达。(3)C2和E2组大鼠心尖部取血离心后,通过ELISA法检测大鼠血清心肌肌钙蛋白T、心肌肌钙蛋白Ⅰ和脑钠肽表达的变化；取出大鼠心脏匀浆后,通过ELISA法检测大鼠心脏Akt和mTOR蛋白的表达。(4)使用HE染色观察C1和El组大鼠心脏组织形态的一般特征。(5)通过免疫组织化学法结合计算机图像处理技术测定C1和E1组大鼠心脏Akt、mTOR、p70S6K和eIF4E的免疫反应阳性面积和光密度。结果：8周中等强度耐力训练后,(1)E1和E2组大鼠较C1组和C2组大鼠,体重发生显著性下降,心脏质量下降,但心肌细胞发生肥大,心系数显著增加。(2)E3组大鼠心脏局部血管紧张素Ⅱ的mRNA表达显著增加,而其1型受体的mRNA表达显著下调；PI3K、PDK1、Akt和AMPK的mRNA表达未见显著增加,mTOR、p70S6K和eIF4E的mRNA表达显著增加。(3)E2组大鼠血清心肌肌钙蛋白T、血清心肌肌钙蛋白Ⅰ、血清BNP和心脏Akt蛋白的含量未发生显著性变化,心脏mTOR的蛋白表达显著增加。(4)E1组大鼠心脏组织HE染色未见病理性改变。(5)心脏Akt蛋白免疫反应阳性面积和光密度未见显著增加；心脏mTOR蛋白、心脏p70S6K蛋白和心脏eIF4E蛋白免疫阳性面积和光密度显著增加。结论：(1)8周中等强度跑台耐力训练可以建立耐力型运动心脏动物模型,为研究耐力训练所致的心肌肥大提供实验基础。耐力型运动心脏重塑后的形态机能特征主要表现在：心肌细胞肥大,心系数增加,心脏泵血功能提高。(2)8周中等强度跑台耐力训练后心肌肌钙蛋白含量未见显著性增高,表明心肌细胞未发生不可逆的坏死, BNP表达未发生显著性变化表明心功未发生恶化,未出现向病理性心肌肥大乃至慢性心衰的发展。(3)8周中等强度跑台耐力训练后,心肌细胞的AT1R的mRNA表达下调,使心脏局部AngⅡ的促细胞增长作用减弱,启动心肌细胞的保护性调节作用,避免心肌过度肥大而产生病理性改变。使心脏局部AngⅡ收缩冠状血管的作用减弱,增加冠状血管血流量,从而保证心脏在运动中的能量代谢需求。说明受体数量及(或)亲和力的变化才是生理性心肌肥大向病理性心肌肥大发展的关键因素,AngⅡ本身数量的变化并不能反映心肌肥大的性质。表明在中等强度耐力训练所致的生理性心肌肥大信号通路网中,AngⅡ/AT1R信号通路不起主要作用。(4)Akt与mTOR之间并不只是一种简单的上下游关系,在心肌细胞处于正常的生理状况下,Akt应是mTOR的上游正性调节因子,而在耐力训练这种特殊的刺激存在时,这种关系受到抑制,运动刺激可直接激活或通过其它途径激活mTOR及其下游因子。(5)8周中等强度的耐力训练后,大鼠心脏PI3K/PDK1/Akt信号通路未被激活或处于一种不完全激活状态。中等强度耐力训练导致的耐力型运动心脏的肥大效应主要是由mTOR/p70S6K/eIF4E信号通路完成的。与正常生理发育导致的心肌肥大不同,PI3K/PDK1/Akt信号通路中的各信号分子并未被激活,表明中等强度耐力训练所致的心脏“生理性肥大”与正常生理发育所致的心脏“生理性肥大”在信号通路的特征上存在差异。更多还原

【Abstract】 In recent years, using the physical activity to prevent and heal the cardiovascular disease becomes a research hotspot. It is reported although there are many positive factors of the cardiac hypertrophy induced by the moderate-intensity endurance exercise; the athlete will still be arrhythmia, cardiac conduction disorders and so on. So the cardiac hypertrophy induced by the moderate-intensity endurance exercise should be physiological cardiac hypertrophy or pathological cardiac hypertrophy; the expression of the representative signaling pathway of physiological cardiac hypertrophy:PI3K/Akt/mTOR signaling pathway, AngⅡand AT1R signaling pathway and other related signaling molecules also has not been reported systematically and clearly.Objective:Using the 8 weeks of moderate-intensity treadmill endurance training, to build a cardiac hypertrophy model of rats. On this basis:According to the expression change of cardiac injury markers (cardiac troponin T, cardiac troponin I and BNP), combined with the histological phenotype, to determine the character of the cardiac hypertrophy induced by moderate-intensity endurance exercise. By the mRNA expression changes of the heart local AngⅡand AT1R, combined with our previous study, to determine whether this representative signaling pathway of pathological cardiac hypertrophy be activated, and the effect of this signaling pathway in cardiac hypertrophy at this time. We want to discuss the effect of moderate-intensity endurance exercise on the expression of cardiac PI3K/Akt/mTOR signaling pathway, clarify the mechanism of the signaling pathway specific changes. So we can determine the character of the cardiac hypertrophy induced by moderate-intensity endurance and provide the morphology and biochemistry theory for the athlete’s heart endocrine and signaling pathway research. Also, we can provide the newer theory and experiment foundation for optimal sports program, effective prevention of cardiac injury, heart health promotion, and exercise prescription for cardiovascular disease. Methods:(1) Using the 8 weeks of moderate-intensity treadmill endurance training, to build a cardiac hypertrophy model of rats. Endurance training programs is accordant with Fenning, combined with the maximum oxygen uptake control method of Wisl?ff, and the relative intensity remain at about 60%-65% VO 2max.(2) Using the real-time quantitative PCR method to test the mRNA expression of PI3K, PDK1, Akt, mTOR, p70S6K, eIF4E, AMPK, AngⅡand AT1R.(3) After anesthetized, removed the blood and heart, testing the levels of serum cardiac troponin T, cardiac troponin I, BNP, cardiac Akt and mTOR by ELISA method. (4) Using the immunohistochemistry combined with computer image processing technology measured the immune response positive area and optical density of Akt, mTOR, p70S6K and eIF4E. (5) Using the HE stain to observe the cardiac morphology.Results:After the cardiac hypertrophy induced by the moderate-intensity endurance exercise, heart function was significantly improved, the levels of serum cardiac troponin T, cardiac troponin I and BNP had not changed significantly; After the moderate-intensity endurance exercise, the mRNA expression of heart local AngⅡwas significantly increased, while AT1R was significantly reduced; After the moderate-intensity endurance exercise, the mRNA expression of PI3K, PDK1, Akt and AMPK were not significantly increased, by using immunohistochemistry combined with computer image processing technology, Akt immune-positive area and optical density were not significantly enhanced, and the immune response did not occur in the cell membrane, the Akt protein content was not significantly increased by ELISA testing. After the moderate-intensity endurance exercise, the mRNA expression of mTOR, p70S6K and eIF4E was increased significantly; by using immunohistochemistry combined with computer image processing technology, the mTOR, p70S6K and eIF4E immune-positive area and optical density were significantly enhanced; the mTOR protein content was significantly increased by ELISA testing. Conclusion:The 8 weeks moderate-intensity treadmill endurance exercise can build an animal cardiac hypertrophy model to provide an experimental basis for studying the athlete’s heart; After the moderate-intensity treadmill endurance training, the serum cardiac troponin slightly elevated, but this could not show the myocardial cell necrosis, and the reason of the routine increase release after exercise may be:after cardiac hypertrophy, cardiac troponin synthesis increased, and cardiac function demand increased. BNP level did not change prompted that the cardiac function did not deteriorating, did not trend to pathological cardiac hypertrophy even the chronic heart failure and other cardiac disease; After the 8 weeks of moderate-intensity treadmill endurance training, the expression of AT1R decreased, so weakened the effect of promoting cell growth by local AngⅡ, then it started the regulation mechanism to prevent cardiac hypertrophy trend to pathological changes. This results suggested that the receptor level and (or) affinity changes was the key factor in the tendency of physiological cardiac hypertrophy to pathological cardiac hypertrophy. The AngⅡlevel and (or) affinity changes did not reflect the character of cardiac hypertrophy. It also shows that the AngⅡ/AT1R signaling pathway is not the key role in the physiological cardiac hypertrophy signaling network, which is induced by endurance training, the key signaling pathway is mTOR signaling pathway. Different With pure physiological cardiac hypertrophy, the PI3K/Akt signaling pathway was not activated, suggested that there are differences between exercise-induced "physiological hypertrophy" and simple "physiological hypertrophy".更多还原