内质网应激相关因子对不同心脏手术心肌保护作用的对比研究

发布时间：2018-05-19 21:15

本文选题：Caspase-12 + NF-kB　；参考：《广西医科大学》2015年硕士论文

【摘要】：目的：在心脏外科的研究中,心肌保护一直是研究的热点,心肌缺血再灌注损伤(MIRI)是在术中导致心肌细胞损伤的最主要因素,也是主要的研究方向。MIRI是一个多因素的病理生理过程,目前已知的通路主要有钙离子超载、线粒体途径、微血管创伤、炎症反应以及脂类物质超氧化等机制,在此研究之上,近年来发现内质网应激(ERS)可以影响细胞凋亡及组织的炎症反应并且在MIRI中发挥重要作用。在ERS中内质网通过激活未折叠蛋白反应(UPR)以保护由ERS所引起的细胞损伤,恢复细胞功能。葡萄糖调节蛋白78(GRP78)可以促进内质网内蛋白折叠、恢复内质网的功能,起到保护细胞的作用,同时也是内质网应激启动的信号,与介导凋亡途径的跨膜蛋白结合,使其处于失活状态。Caspase-12位于内质网胞浆面,该物质是内质网应激介导凋亡的一种特异性介质,仅仅存在于内质网应激中,并介通过激活Caspase-3介导凋亡。核因子-κB(NF-κB)是一个多向性核转录调节因子,在多种急性应激反应中表达,目前被认为是内质网应激介导的炎症反应的关键因子,关于其的损伤心肌作用和心肌保护作用均有报道,上述三种蛋白及调控因子具体作用机制复杂且先前的研究多在细胞层面进行,关于动物实验亦或人体中的实验目前尚待进一步研究。对于减少术中心肌损伤,体外循环下心脏不停跳手术有独到的优势,其术式在90年代末开始开展应用,并逐渐发展,已有了长足的进步,关于该术式的研究也充分证明了其有良好的预防MIRI作用,可以有效的进行脑、肺、肾脏等多脏器的保护。但关于其分子层面的研究目前尚少。本实验建立体外循环的模型,探索心脏停跳及不停跳手术的情况下,ERS的反应强度及细胞凋亡程度,探索ERS以及其中的三种调节因子在不同手术方式下的表达情况,并对比手术的优缺点。方法：动物由广西医科大学动物实验中心提供,为成年新西兰大白兔共36只,将实验动物随机分为停跳组(体外循环心脏停跳组,n=12),不停跳组(体外循环心脏不停跳组,n=12)以及对照组(假手术组)。建立兔体外循环模型。假手术组仅作开关胸手术,不做其他处理。不停跳组体外通过颈总动脉作为动脉灌注,颈总静脉作为静脉引流,进而建立循环,维持体外循环约120min,整个实验过程中不进行停跳液的灌注、不阻断升主动脉及上下腔静脉、冠状动脉维持血流灌注。停跳组建立体外循环后,并行循环约15min,将实验动物温度降低至28℃～30℃时将主动脉阻断,并从主动脉根部灌注St. Thomas Ⅱ停搏液(15ml/kg,约4℃),总共灌注3次St. Thomas Ⅱ停搏液(后两次分别于30min和60min分别进行,灌注量为第一次的一半)。灌注完毕后缓慢复温,于实验动物处于34℃时基本恢复全流量,最后使心脏复跳并行体外循环。实验动物生命体征平稳后停止体外循环操作。实验成功的标志为实验动物在停止体外循环1个小时后仍存活,处死实验动物,进行心肌标本的采集。根据免疫组化试剂盒的步骤分别对三组实验动物心肌进行GRP78、Caspase-12以及NF-κB的表达检测,并对三组动物的心肌行TUNEL法检测凋亡率。结果：(1)GRP78的表达量：停跳组(72.24%±7.89%),不停跳组(16.53%±2.59%),假手术组(1.68%±1.19%),组间差别有明显统计学意义(P0.01)。(2) NF-κB的表达量：停跳组(73.70%±6.70%),不停跳组(13.81%±1.79%),假手术组(2.42%±1.42%),组间差别有明显统计学意义(P0.01)(3)Caspase-12的表达量：停跳组(73.74%±7.15%),不停跳组(14.49%±2.29%),假手术组(2.46%±1.56%),组间差别有明显统计学意义(P0.01)(4)心肌细胞凋亡指数(AI)：心脏停跳组凋亡率为(10.11±0.53),心脏不停跳组(4.27±0.61),对照组为(1.33±0.35)(P0.01)。组间差别有统计学意义(P0.01)(5)相关性分析：心脏停跳组以及心脏不停跳组心肌细胞凋亡率与该组caspase-12表达量相关系数分别为为0.961(P0.01)以及0.912(P0.01),呈明显的正相关；与该组GRP78表达量相关系数分别为0.827(P0.01)以及0.831(P0.01),呈正相关,与该组NF-κB表达量相关系数分别为0.317(P0.05)以及0.281(P0.05),无明显相关性；结论：(1)GRP78在心脏停跳及不停跳组中都明显升高,说明内质网应激参与了体外循环中细胞的损伤。(2)Caspase-12在两个实验组中明显升高说明了体外循环中心肌细胞损伤明显有心肌凋亡参与(3) NF-κB在两组中表达明显上调说明NF-κB通路参与了体外循环下的心肌内质网应激及相关炎症反应,其与心肌凋亡率相关性不明显可以佐证其具有抗凋亡和促双重作用。(4)心脏不停跳手术可以减少内质网应激,减少心肌损伤,保护心功能。
[Abstract]:Objective: in the study of cardiac surgery, myocardial protection has always been a hot spot of research. Myocardial ischemia reperfusion injury (MIRI) is the most important factor in the injury of cardiac myocytes in the operation, and the main research direction.MIRI is a multi factor pathophysiological process. The main known pathways are calcium overload, mitochondrial pathway, and microsatellite pathway at present. In recent years, endoplasmic reticulum stress (ERS) can affect apoptosis and tissue inflammation and play an important role in MIRI. In ERS, endoplasmic reticulum (endoplasmic reticulum) can protect cell damage caused by ERS by activating unfolded protein reaction (UPR). The function of glucose regulating protein 78 (GRP78) can promote the internal protein folding in the endoplasmic reticulum, restore the function of endoplasmic reticulum, play a role in protecting the cells, and also signal the initiation of endoplasmic reticulum stress, combined with the transmembrane protein which mediates the apoptosis pathway, and make it inactive.Caspase-12 in the endoplasmic reticulum, and the substance is within. The mesoplasmic reticulum stress mediated apoptosis is a specific medium that only exists in endoplasmic reticulum stress and mediates apoptosis through activation of Caspase-3. Nuclear factor kappa B (NF- kappa B) is a multidirectional nuclear regulatory factor, expressed in a variety of acute stress responses, and is currently considered to be a key factor in the inflammatory response mediated by endoplasmic reticulum stress. There are reports of myocardial injury and myocardial protection. The specific mechanisms of these three proteins and regulatory factors are complex and previous studies are mostly at the cellular level. Animal experiments and human experiments are still to be further studied. For the reduction of cardiac muscle injury, cardiac arrest under cardiopulmonary bypass is performed. The unique advantage, its operation began to carry out the application in the late 90s, and gradually developed, has made great progress. The study of this type of operation has also fully proved that it has good preventive effect on MIRI and can effectively protect many organs such as brain, lung, kidney and so on. However, there are few studies on its molecular level. This experiment is established in vitro. To explore the reaction intensity and the degree of apoptosis of ERS, explore the expression of ERS and the three regulatory factors under different surgical methods, and compare the advantages and disadvantages of the operation. Methods: the animals were provided by the animal experiment center of Guangxi Medical University for adult New Zealand. A total of 36 white rabbits were randomly divided into stop jump group (cardiopulmonary bypass cardiac arrest group, n=12), non stop jump group (extracorporeal circulation heart beating group, n=12) and control group (sham operation group). The rabbit extracorporeal circulation model was established. The sham operation group was only operated on the switch chest operation and did not do other treatment. As a venous drainage, the total neck vein was used as a venous drainage, and the circulation was established to maintain an external circulation of about 120min. During the whole experiment, the blood perfusion was not carried out, the ascending aorta and the superior and inferior vena cava were not blocked. The coronary artery was maintained by the coronary artery. After the suspension of the external cycle, the parallel circulation was about 15min, and the temperature of the experimental animals was reduced to 28 to 30. The aorta was blocked, and the St. Thomas II cardioplegia (15ml/kg, about 4 C) was perfused from the root of the aorta, and 3 St. Thomas II cardioplegia was perfused (two times respectively in 30min and 60min, respectively, half of the first time respectively). After the perfusion was completed, the temperature was slowly rewarming, and the whole flow was basically recovered at 34 C, and the heart was finally made to the heart. The sign of the experiment was that the experimental animals were still alive after 1 hours of cessation of cardiopulmonary bypass. The experimental animals were killed and the myocardial specimens were collected. The GRP78 of the three groups of experimental animals was carried out according to the steps of the immunohistochemical kits, Caspa The expression of se-12 and NF- kappa B was detected, and TUNEL method was used to detect the apoptosis rate of three groups of animals. Results: (1) the expression of GRP78: (72.24% + 7.89%), non stop group (16.53% + 2.59%), sham operation group (1.68% + 1.19%), and there was significant difference between groups (P0.01). (2) the expression of NF- kappa B (73.70% + 6.70%), ceaselessly Jump group (13.81% + 1.79%), sham operation group (2.42% + 1.42%), there was significant difference between groups (P0.01) (3) Caspase-12 expression: stop jump group (73.74% + 7.15%), non stop jump group (14.49% + 2.29%), sham operation group (2.46% + 1.56%), there was significant difference between groups (P0.01) (4) myocardial apoptosis index (AI): cardiac arrest group apoptosis The rate was (10.11 + 0.53), the heart beating group was (4.27 + 0.61) and the control group was (1.33 + 0.35) (P0.01). The difference between the groups was statistically significant (P0.01) (5) correlation analysis: the myocardial apoptosis rate of cardiac arrest group and heart beating group was 0.961 (P0.01) and 0.912 (P0.01), respectively, and was obviously positive. Correlation: the correlation coefficient of GRP78 expression was 0.827 (P0.01) and 0.831 (P0.01), respectively, and the correlation coefficient of NF- kappa B expression was 0.317 (P0.05) and 0.281 (P0.05), respectively. Conclusion: (1) GRP78 was significantly increased in the cardiac arrest and non stop jump groups, indicating that endoplasmic reticulum stress was involved in the in vitro development. (2) the significant increase of Caspase-12 in the two experimental groups indicated that myocyte injury in cardiopulmonary bypass was significantly associated with myocardial apoptosis (3) the expression of NF- kappa B in the two groups indicated that NF- kappa B pathway was involved in cardiac endoplasmic reticulum stress and related inflammatory responses under cardiopulmonary bypass, which was not related to the rate of myocardial apoptosis. (4) beating heart surgery can reduce endoplasmic reticulum stress, reduce myocardial injury and protect heart function.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.2

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