卡维地洛对缺氧复氧诱导的H9C2心肌细胞凋亡的保护作用及机制

发布时间：2018-06-07 03:02

本文选题：H9C2心肌细胞株 + 凋亡　；参考：《安徽医科大学》2016年博士论文

【摘要】：研究背景与目的:缺血再灌注损伤是在血栓性疾病中常见的现象。缺血心肌在得到再灌注的同时,由于激活免疫反应而导致梗塞周围出现更多的损伤,并与心肌细胞凋亡的增加有关。损伤的结果会导致心肌细胞的丢失和梗塞面积的扩大。因此,研究缺血再灌注损伤所致的心肌细胞凋亡具有重要的临床意义。有多种信号传导通路参与心肌细胞的凋亡,TLR4-NF-κB通路在其中起着重要的作用。TLR4是TOLL系列受体之一,能被多种刺激信号激活,如应激、炎症、坏死等。TLR4主要在心脏表达,将信号传导至胞内并在酶的作用下活化NF-κB,而NF-κB则进入核内引起相关基因的表达,促进炎症因子的产生。β-arrestin被认为是一种多功能受体蛋白,参与GPCR的脱敏。β-arrestin通过结合NF-κB的抑制因子IкBα而调控NF-κB基因的表达。因此,β-arrestin被认为是TLR4-NF-κB信号通路的负调控因子。卡维地洛是一种新型的非选择性β受体阻滞剂,应用于治疗高血压和心绞痛。在慢性心力衰竭患者中,卡维地洛能够降低其住院率和死亡率。在多项临床研究中,相对于其他β受体阻滞剂,卡维地洛在抑制左室重构和保持射血分数方面显示出显著的优势。这种作用涉及多种机制,抑制心肌细胞凋亡是其中之一。然而,有关卡维地洛对TLR4-NF-κB介导的心肌细胞凋亡的影响尚不清楚。卡维地洛是否能对β-arrestin产生影响更有少见报道。因此,我们准备两部分实验来探讨卡维地洛对缺血再灌注损伤心肌细胞模型中TLR4-NF-κB通路的作用以及β-arrestin表达的影响。材料与方法:第一部分H9C2心肌细胞株的培养和缺血/再灌注模型建立方法:培养H9C2心肌细胞株,采用缺氧复氧模型模拟心肌缺血再灌注损伤,缺氧4小时后按照复氧时间不同分为5组:对照组,复氧4h组,复氧6h组,复氧10h组。观察指标包括荧光显微镜下细胞生长形态变化,mtt值,培养液中ldh、mda及sod含量。结果:H9C2心肌细胞株在体外培养成功,并建立缺氧复氧模型。缺氧复氧后细胞形态镜下发生明显变化,表现为核染色细胞增多。在实验组中,mtt值显著下降,并随着复氧时间延长而更为显著。ldh和mda含量分别显著增加,sod则显著降低,具有显著性差异。结论:本研究在体外通过缺氧复氧而成功构建H9C2心肌细胞模拟缺血再灌注模型,缺氧4小时复氧6小时可造成显著心肌细胞损伤,为进一步实验提供标本。第二部分H9C2心肌细胞凋亡中tlr4-nfκb、β-arrestin的表达和卡维地洛的作用方法:采用不同剂量卡维地洛(1um、5um、10umol/l)、tlr4封闭抗体(20ug/ml)、nf-κb抑制剂pdtc(100umol/l)预孵育H9C2心肌细胞1h后,建立模拟缺血再灌注模型。实验分为7组:正常对照组(control组),模拟缺血再灌注组(sd组),小剂量组卡维地洛组(car1卡维地洛1umol/l),中剂量组卡维地洛组(car5卡维地洛5umol/l),高剂量组卡维地洛组(car10卡维地洛10umol/l),tlr4封闭抗体组(tlr4-ab组tlr4封闭抗体20ug/ml),nf-κb抑制剂pdtc组(pdtc组pdtc100umol/l)。应用蛋白质印迹技术检测细胞凋亡蛋白bax,bcl-2。通过荧光定量pcr法测定tlr4、核蛋白nf-κb、β-arrestin的表达。结果:H9C2心肌细胞株在i/r后,与正常对照组相比,tunnel检测细胞凋亡率增高,bax升高,bcl-2下降,tlr4和nf-κb表达增多,β-arrestin表达减少。在经过卡维地洛预处理的H9C2i/r模型中,与i/r组相比bcl-2明显增加,细胞凋亡率和bax显著降低。通过rt-pcr技术检测,在卡维地洛预处理组中,tlr4和nf-κb表达量减少,β-arrestin表达增多。上述指标的变化,在中、高剂量卡维地洛组中更为显著。tlr4抗体和nf-κb阻断剂(pdtc)预处理H9C2心肌细胞,与i/r组相比,可表现出与卡维地洛组相同的显著改变。结论:1.TLR4-NF-κB信号通路与H9C2心肌细胞株I/R所致凋亡过程有关2.卡维地洛可以通过抑制TLR4/NF-κB表达而减轻H9C2心肌细胞I/R所致的凋亡,在中、高剂量组中更为显著3.卡维地洛可能会激活β-arrestin,负调控TLR4-NF-κB信号。
[Abstract]:Background and purpose: ischemia-reperfusion injury is a common phenomenon in thrombotic diseases. Ischemic myocardium is involved in reperfusion and causes more damage around the infarct due to the activation of the immune response. It is related to the increase of apoptosis of myocardial cells. The result of injury will lead to the loss of myocardial cells and the enlargement of infarct size. Therefore, the study of myocardial apoptosis induced by ischemia-reperfusion injury is of important clinical significance. There are a variety of signal transduction pathways involved in the apoptosis of cardiac myocytes. TLR4-NF- kappa B pathway plays an important role in.TLR4, one of the TOLL series receptors, which can be activated by a variety of stimuli, such as stress, inflammation, and necrosis, mainly in the heart. Dirty expression, transmit the signal to the intracellular and activate NF- kappa B under the action of enzyme, while NF- kappa B enters the nucleus to induce the expression of related genes and promote the production of inflammatory factors. Beta -arrestin is considered to be a kind of multifunctional receptor protein and participates in desensitization of GPCR. The expression of beta -arrestin regulates the expression of NF- kappa gene by binding NF- kappa B inhibitor I B alpha. Therefore, beta -arrestin is considered to be a negative regulator of the TLR4-NF- kappa B signaling pathway. Carvedilol is a new non selective beta blocker, used in the treatment of hypertension and angina. In patients with chronic heart failure, carvedilol can reduce the rate of hospitalization and mortality. In a number of clinical studies, it is relative to other beta receptors. The effect of carvedilol on TLR4-NF- kappa B induced cardiomyocyte apoptosis is not clear. Whether carvedilol can produce a shadow of beta -arrestin, however, is not clear about the effect of carvedilol on the apoptosis of cardiac myocytes. There are more rare reports. Therefore, we prepare two experiments to explore the effect of carvedilol on the TLR4-NF- kappa B pathway in the myocardial cell model of ischemia-reperfusion injury and the effect of the expression of beta -arrestin. Materials and methods: the first part of the culture of H9C2 cardiomyocytes and the establishment of ischemia / reperfusion model: the cultivation of H9C2 cardiac myocytes. A hypoxic reoxygenation model was used to simulate myocardial ischemia reperfusion injury, and 4 hours after hypoxia were divided into 5 groups according to the time of reoxygenation: control group, reoxygenation 4H group, reoxygenation 6h group and reoxygenation 10h group. The observation indexes include the changes of cell growth morphology under fluorescence microscope, MTT value, LDH, MDA and SOD content in the culture medium. Results: H9C2 cardiac myocytes were cultured in vitro. In the experimental group, the MTT value decreased significantly, and the content of.Ldh and MDA increased significantly with the prolonged reoxygenation time, and the sod decreased significantly. Conclusion: This study was in vitro. The model of H9C2 myocardial ischemia reperfusion was successfully constructed by hypoxia and reoxygenation. 4 hours anoxic reoxygenation for 6 hours could cause significant myocardial damage and provide specimens for further experiments. Second part of H9C2 cardiomyocyte apoptosis, the expression of tlr4-nf kappa B, the expression of beta -arrestin and the action method of Carvedilol (1 Um, 5um, 10umol/l), TLR4 closed antibody (20ug/ml), nf- kappa B inhibitor PDTC (100umol/l) preincubated H9C2 myocardial cells 1H, the model of simulated ischemia reperfusion was established. The experiment was divided into 7 groups: normal control group (control group), simulated ischemia reperfusion group, carvedilol group (carvedilol) and medium dose group carvedilol group. 5 carvedilol 5umol/l), high dose carvedilol group (car10 carvedilol 10umol/l), TLR4 closed antibody group (tlr4-ab group TLR4 closed antibody 20ug/ml), nf- kappa B inhibitor PDTC group (PDTC group pdtc100umol/l). N expression. Results: compared with the normal control group, the apoptosis rate of H9C2 cells increased, Bax increased, bcl-2 decreased, TLR4 and nf- kappa B expression increased, and the expression of beta -arrestin decreased. In the H9C2i/r model pretreated by carvedilol, the rate of apoptosis was significantly increased and the rate of apoptosis decreased significantly. After RT-PCR test, the expression of TLR4 and nf- kappa B decreased and the expression of beta -arrestin increased in the carvedilol preconditioning group. In the high dose carvedilol group, a more significant.Tlr4 antibody and nf- kappa B blocker (PDTC) pretreated the H9C2 cardiomyocytes in the high dose carvedilol group. Compared with the I /r group, the same significant changes were shown in the carvedilol group. Conclusion: 1.TLR4-NF- kappa B signaling pathway is related to the apoptosis process induced by H9C2 myocardial cell line I/R. 2. carvedilol can reduce the apoptosis of I/R induced by H9C2 cardiomyocytes by inhibiting the expression of TLR4/NF- kappa B. In the high dose group, more significantly 3. carvedilol may activate beta -arrestin and negatively regulate TLR4-NF- kappa B signal.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54

【参考文献】