【作者】 雷泽源；

【导师】 黄跃生；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 烧伤后早期可迅速发生心肌损害。由于心脏的特殊重要性,其损害不仅引起心功能不全,还是诱发或加重休克的重要因素,我们称这一现象为“休克心”。阐明烧伤后迅即发生心肌损害和功能减退的机制,对防治心肌损害和烧伤休克治疗都有重要临床意义。心脏局部存在有独立的肾素-血管紧张素系统(rennin angiotensin system,RAS),可身合成、释放肾素、血管紧张素。与循环RAS不同,心脏局部RAS采用自分泌,旁分泌等方式只作用于心脏局部,主要参与心肌局部血流量和血管紧张性和心肌收缩力的调节,在维持心血管正常功能活动及心血管疾病的发生、发展过程中起重要的作用。但该系统是否参与烧伤后迅即发生的心肌缺血损伤,尚无系统的研究报道。本研究观察烧伤早期RAS的主要生物活性成分的变化及其与心肌损伤的影响,同时对RAS进行干预,为揭示烧伤早期心脏损伤的机制和寻找内源性保护措施奠定基础。材料和方法1、建立大鼠30%TBSAⅢ°烫伤模型,动态观察烧伤后24h大鼠心肌组织和血清中RAS活性成分变化,了解烧伤早期RAS的激活情况;并同时观察大鼠心肌力学和血流动力学指标,血清中CTn-I的变化。2、调节RAS系统对烧伤早期大鼠心功能及心肌损害影响:根据第一部分实验选择心脏损伤最重的时相点,利用血管紧张素转化酶抑制剂(ACEi)依那普利拉和Ang(1-7)分别调节ACE-AngII轴和ACE2-Ang(1-7)轴,应用多导信息收集系统观察大鼠心肌力学和血流动力学指标,并检测血清中CTn-I的变化。3、应用大鼠离体心脏Langendorff灌流系统,以大鼠烧伤血清模拟致伤,通过调节RAS系统观察大鼠心肌力学指标,冠脉流量以及冠脉流出液中的肌酸激酶(CK)、乳酸脱氢酶(LDH)的活性。4、原代培养SD大鼠心脏微血管内皮细胞,观察调节RAS系统对烧伤血清刺激下大鼠心脏微血管内皮细胞细胞活力、NO、LDH、SOD等指标的变化。主要结果:1、大鼠严重烧伤后很快出现心肌损害,于6h心肌力学和血流动力学指标(SP、DP、LVSP、LVEDP及±dp/dtmax)达低谷,心肌损伤指标血清CTn-I达峰值,而大鼠心肌组织和血清中ACE和AngII轴于1h开始升高并于6h达到峰值,心肌组织中ACE2变化不明显。2、利用血管紧张素转化酶抑制剂(ACEi)依那普利拉和Ang(1-7)分别调节ACE -AngII轴和ACE2 -Ang(1-7)轴,可使伤后6h心肌力学和血流动力学指标(SP、DP、LVSP、LVEDP及±dp/dtmax)明显提升,心肌损伤指标血清CTn-I明显降低。3、应用烧伤后6h的大鼠血清对大鼠心脏进行Langendorff离体灌流模拟致伤后,离体心脏冠脉流量明显减少,左心功能指标LVSP、±dp/dtmax显著降低,LVEDP显著升高,提示左心功能明显减退;冠脉流出液中CK、LDH的活性亦明显升高,提示出现明显心肌损伤,心肌损伤可能是由于烧伤血清引起RAS激活造成冠脉血流剧减所致。使用依那普利拉和Ang(1-7)可明显增加冠脉流量,降低冠脉流出液中CK、LDH的活性。4、成功培养了原代SD大鼠CMEC并鉴定,烧伤血清刺激12h后,SD大鼠CMEC细胞活力明显下降,培养液中SOD、NO下降,LDH升高。使用含有依那普利拉和Ang(1-7)的烧伤血清可明显提高CMEC细胞活力及培养液中SOD、NO的水平,降低LDH。结论:1、严重烧伤后很快即出现心功能下降和心肌损害,进一步验证了严重烧伤早期缺血缺氧损害的“休克心”学说。2、作为循环的重要调节系统,烧伤早期RAS的ACE-AngII轴迅速激活,于伤后6h达到峰值,并与心肌损害存在时效关系。而ACE2无明显变化,导致了RAS的ACE2-Ang(1-7)轴和ACE-AngII轴失衡,提示RAS的失衡可能是导致烧伤早期心肌缺血损害的重要因素之一。3、在烧伤早期,主动调节ACE2-Ang(1-7)轴和ACE-AngII轴的失衡状态可减轻心肌损害,证实了RAS失衡是导致烧伤早期心肌损害重要因素之一。提示在烧伤早期主动调节ACE2 -Ang(1-7)轴和ACE-AngII轴可能成为减轻烧伤早期心肌缺血损害的重要靶点。4、通过调节RAS可提升烧伤血清离体灌流大鼠心脏的冠脉流量,改善左心功能,减轻心肌损害。提示RAS激活是造成烧伤早期冠脉血流量减少,引发心肌损伤的重要原因。5、烧伤血清刺激可使SD大鼠CMEC细胞活力明显下降,培养液中SOD、NO下降,LDH升高,引发CMEC损伤。通过使用含有依那普利拉和Ang(1-7)的烧伤血清可明显改善CMEC功能。说明烧伤早期ACE- AngⅡ轴的激活不仅可直接影响心脏血流,还可通过导致CMEC功能损伤引起心肌损害。更多还原

【Abstract】 Myocardial damage may occur due to severe burn at early stage. Because of the special importance of the heart, its damage may not only cause cardiac dysfunction, but also induce or exacerbate burn shock. Ischemia/hypoxia is the important factor for myocardial damage during early postburn stage.Local rennin angiotensin system(RAS) existing in heart can synthesize and release rennin angiotensin. Different from circulatory RAS, the local RAS only effects on the heart itself to regulate myocardial regional blood flow, vascular tone and myocardial contractility. However, little is known about the contribution of RAS in myocardial damage during early postburn stage.The purpose of present study is to observe the changes of RAS components and its effects on heart function and myocardial damage, and to reveal the mechanism of heart damage and endogenous protection at early stage postburn by regulating RAS.Material and MethodsBoth in vivo and in vitro experiments were carried out in the study.1. In in vivo study, rats inflicting with 30% TBSA of full-thickness burns were used. The changes of RAS bioactive components in 24h postburn, and hemodynamics, myocardial mechanical indices, serum CTn-I level were investigated.2. Effects on heart function and myocardial damage by using Enalaprilat and Ang(1-7) to regulate the ACE-AngII axis and the ACE2-Ang(1-7) axis were investigated. Hemodynamics, myocardial mechanical indices, serum CTn-I level were assayed.3. An isolated heart Langendorff perfusion system was established. The isolated heart was inflicted with burn sera. Myocardial mechanical indices, coronary flow(CF) and LDH、CK level in coronary flow fluid were detected.4. Primary SD rat cardiac microvascular endothelial cells (CMEC) were cultured , stimulated with burn sera. CMEC vitality, NO, LDH and SOD level in culture fluid were assessed. Results:1. Myocardial damage occurred soon after severe burn, hemodynamics, myocardial mechanical indices (reflected by LVSP、LVEDP and±dp/dtmax) decreased to the lowest at 6h. Serum CTn-I peaked at 6h. ACE–AngII axis in myocardial and serum reased rapidly at 1h, peaked at 6h postburn. However, myocardial ACE2 was not activated obviously .2. After using Enalaprilat and Ang(1-7), the hemodynamics, myocardial mechanical indics including LVSP, LVEDP and±dp/dtmax were improved and serum CTn-I decreased obviously.3. The results of the isolated heart Langendorff perfusion system showed that CF and left cardiac function decreased obviously, and LDH, CK level in coronary flow fluid increased obviously, indicating that decrease of left ventricle function and myocardial damage may cause the decreasing CF after RAS activation. CF increased, and CK, LDH decreased after Enalaprilat and Ang(1-7) treatment.4. Primary CMEC from SD rat was uccessfully cultured. After stimulated by burn sera for 12h, the vitality of CMEC and levels of SOD, NO in culture fluid were decreased, and LDH increased. Cell vitality and level of SOD, NO in culture fluid increased, and LDH decreased by using burn sera containing Enalaprilat and Ang(1-7).Conclusion:1. Heart dysfunction and myocardial damage occurred immediately following severe burns, which further proved the“shock heart”theory of ischemic/hypoxic injury.2. As the important regulating system of circulation, ACE-AngII axis of RAS was rapidly activated at early stage postburn, which showed time-effect relationship with myocardial damage. But ACE2 was not activated, causing imbalance of ACE2-Ang(1-7) axis and ACE-AngII axis. It was indicated the imbalance of RAS may be one of the important reasons for heart damage. Actively regulating ACE2-Ang(1-7) axis and ACE-AngII axis at early stage postburn may be the important target to lessen myocardial ischemic injury.3. Myocardial damage was lessened by initiatively regulating the imbalance of ACE2-Ang(1-7) axis and ACE-AngII axis at early stage of severe burn, which indicated the imbalance of RAS was one of the important reasons for heart damage at early stage postburn. 4. By regulating RAS, CF was increased, left ventricle function was improved and myocardial damage was lessened,which imported that decrease of CF caused by RAS activation was the important reason for heart damage.5. Following burn sera stimulation, vitality of CMEC and level of SOD、NO in culture fluid was decreased, LDH increased. CMEC function was improved after treating with burn sera containing Enalaprilat and Ang(1-7). It is concluded that RAS activation led to heart damage at early stage postburn not only by affecting heart perfusion but also function of endothelial cell.更多还原