曲美他嗪对离体心肌细胞缺血再灌注损伤诱导的自噬基因Beclin1的影响

发布时间：2018-05-18 20:21

本文选题：缺血再灌注损伤 + 自噬　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:冠心病(coronary heart disease,CHD)是心血管疾病中引起死亡的首要原因。随着人们生活水平的提高,冠心病患者人数逐年上升,急性心肌梗死(acute myocardial infarction,AMI)患者也明显增多。目前临床上治疗急性心肌梗死主要采用溶栓、经皮冠状动脉介入治疗术(percutaneous coronary intervention,PCI)以及冠状动脉旁路移植术(coronary artery bypass grafting,CABG)等手段以开通阻塞血管,挽救濒死心肌,但随之而来的缺血再灌注(ischemia reperfusion,IR)损伤却严重影响治疗效果及患者预后,因此对于缺血再灌注损伤机制的研究越来越受重视。心肌缺血再灌注损伤涉及多种机制,包括氧自由基增多、钙超载、炎症反应、线粒体损伤、细胞凋亡等。近年来的研究表明,自噬是一种新的程序性死亡(II型程序性死亡)方式,成为新的研究热点,并且被认为在心肌缺血再灌注损伤中发挥着重要作用。自噬是一个在进化上高度保守的受损或功能障碍的蛋白质或细胞器降解的过程。适度的自噬对机体起保护作用,过度的自噬则会加剧细胞死亡。研究表明,自噬在缺血再灌注损伤中表现出“双刃剑”的作用,缺血期主要起保护作用,而再灌注期自噬过度激活则表现出损伤作用,可导致细胞死亡。自噬的发生非常复杂,其中自噬体的形成是自噬发生的关键环节。自噬由大多数自噬基因(autophagy-related gene,Atg)进行调控,其中Beclin1基因是酵母Atg6的同系物,是形成自噬体的必需分子,可介导自噬相关基因定位于自噬体膜,并与多种蛋白反应调控自噬体的形成与成熟。因此抑制Beclin1的表达可有效阻断自噬的发生。近年来心肌能量代谢药物通过联合常规改善血流药物(硝酸酯类、β-受体阻滞剂、钙拮抗剂等)治疗心肌缺血备受关注,随着心肌能量代谢研究的不断深入,曲美他嗪优化心肌代谢的作用机制成为新的研究热点。曲美他嗪(trimetazidine,TMZ)作为调节心肌能量代谢的药物,通过部分抑制游离脂肪酸的氧化,减少氧自由基产生,减轻钙超载,发挥线粒体保护、抗氧化应激、抗细胞凋亡等多方面的药理作用,维持ATP的产生及缺血心肌细胞的能量代谢功能,从而发挥对心肌的保护作用。但其对自噬的影响目前文献报道较少。因此,本实验以体外培养乳鼠心肌细胞为研究及观察对象,利用离体心肌细胞缺氧复氧(hypoxia reoxygenation,HR)模型模拟心肌缺血再灌注损伤,并给予不同浓度的曲美他嗪干预。比较各组心肌细胞活力、培养上清液LDH含量以评价各组心肌细胞的受损情况及曲美他嗪对心肌缺血再灌注损伤的保护作用;采用RT-PCR检测自噬基因Beclin 1 m RNA水平,以观察自噬基因Beclin1在缺血再灌注损伤的表达情况及探讨曲美他嗪对自噬基因Beclin1的影响,为临床用药提供新思路及依据。方法:出生2-3天的健康Sprague Dawley(SD)乳鼠,来自河北医科大学动物中心,提取并培养心肌细胞,并将心肌细胞随机分为5组:1)正常对照组(A组):含10%胎牛血清DMEM培养基(37℃、5%CO2、95%空气)培养26.5h;2)HR模型组(B组):不含血清的低糖DMEM培养基培养30min后,三气培养箱(92%N2、5%CO2、3%O2)中培养24h,然后在含10%胎牛血清DMEM培养基(37℃、5%CO2、95%空气)培养2h模拟缺血再灌注损伤;3)0.2μmol/L曲美他嗪预处理组(C组):含0.2μmol/L曲美他嗪,不含血清的低糖DMEM培养基培养30min后,建立HR模型,模拟缺血再灌注(同B组);4)1.0μmol/L曲美他嗪预处理组(D组):含1.0μmol/L曲美他嗪,不含血清的低糖DMEM培养基培养30min后,建立HR模型,模拟缺血再灌注(同B组);5)5.0μmol/L曲美他嗪预处理组(E组):含5.0μmol/L曲美他嗪,不含血清的低糖DMEM培养基培养30min后,建立HR模型,模拟缺血再灌注(同B组);各组细胞处理后,应用噻唑蓝(MTT)比色法检测心肌细胞活力、全自动生化分析仪检测培养清乳酸脱氢酶(LDH)含量、采用RT-PCR技术测定各组自噬基因Beclin1 m RNA表达水平。结果:1心肌细胞活力结果:B组心肌细胞活力为A组的61.26±6.30%(P0.05),C组、D组、E组心肌细胞活力分别为A组的70.34±7.17%、79.33±6.90%、89.43±6.02%,均明显高于B组(P0.05),其中E组高于C组、D组(P0.05);2乳酸脱氢酶(LDH)测定结果:B组、C组、D组、E组LDH含量分别为176.96±12.70 U/L、157.87±13.38 U/L、126.44±12.41U/L、97.13±12.14U/L均明显高于A组的65.18±16.99U/L(P0.05),C组、D组、E组LDH含量均低于B组(P0.05),E组低于C组、D组(P0.05),与心肌细胞活力结果共同说明通过HR建立的离体IR模型是有效的,HR造成离体心肌细胞损伤,而曲美他嗪能减轻HR诱导的心肌细胞损伤,并具有一定的浓度依赖性;3 RT-PCR结果:B组心肌细胞自噬基因Beclin1 m RNA相对表达量明显高于A组,达A组的2.99±0.19倍(P0.05),C组、D组、E组心肌细胞自噬基因Beclin1 m RNA相对表达量分别为(2.65±0.24、2.19±0.17、1.64±0.20)均低于B组(P0.05),尤以E组明显,说明HR可诱导心肌细胞自噬基因Beclin1 m RNA水平增加,而曲美他嗪能抑制自噬基因Beclin1 m RNA的表达,并具有一定的浓度依赖性。结论:1本实验通过培养乳鼠心肌细胞给予低糖、缺氧后复糖、复氧条件,能明显降低心肌细胞活力,增加LDH含量,有效模拟缺血再灌注模型。2曲美他嗪能减轻缺血再灌注引起的心肌细胞损伤,并具有一定的浓度依赖性。3缺血再灌注损伤可诱导心肌细胞自噬基因Beclin1 m RNA水平增加。4曲美他嗪可减少缺血再灌注诱导的心肌细胞自噬基因Beclin1m RNA的表达,并具有一定的浓度依赖性。
[Abstract]:Objective: coronary heart disease (CHD) is the leading cause of death in cardiovascular disease. With the improvement of people's living standard, the number of patients with coronary heart disease (acute myocardial infarction, AMI) is also increasing, and the patients with acute myocardial infarction (acute, infarction, AMI) are also increasing. The main clinical treatment of acute myocardial infarction is thrombolytic and percutaneous coronary intervention. Percutaneous coronary intervention (PCI) and coronary artery bypass grafting (coronary artery bypass grafting, CABG) are used to open blocking vessels to save the dying myocardium, but the attendant ischemic reperfusion (ischemia reperfusion, IR) seriously affects the treatment effect and the prognosis of the patients. The mechanism of ischemia-reperfusion injury is becoming more and more important. Myocardial ischemia reperfusion injury involves a variety of mechanisms, including the increase of oxygen free radicals, calcium overload, inflammatory reaction, mitochondrial damage, cell apoptosis and so on. Recent studies have shown that autophagy is a new procedure of programmed death (II type programmed death) and has become a new study. Hot spots and considered to play an important role in myocardial ischemia reperfusion injury. Autophagy is an evolutionary highly conserved damaged or dysfunctional protein or organelle degradation process. Moderate autophagy protects the body, excessive autophagy exacerbates cell death. Autophagy is found to be in ischemia reperfusion. The role of "double-edged sword" is shown in the injury, and the ischemic period mainly plays a protective role, and the excessive activation of autophagy in the reperfusion period shows the damage effect, which can lead to cell death. Autophagy is very complex, in which autophagy is the key link of autophagy. Autophagy is made by most autophagy (autophagy-related gene, Atg). The Beclin1 gene is the homologue of yeast Atg6, which is essential for the formation of autophago, which mediates autophagic related genes to be located in the autophagic membrane, and regulates the formation and maturation of autophagic bodies with a variety of proteins. Therefore, inhibition of the expression of Beclin1 can effectively block the occurrence of autophagy. Routine improvement of blood flow drugs (nitrates, beta blockers, calcium antagonists, etc.) is concerned about the treatment of myocardial ischemia. With the continuous development of the study of myocardial energy metabolism, the mechanism of trimetazidine in the optimization of myocardial metabolism has become a new research hotspot. Trimetazidine (TMZ) is used as a drug to regulate cardiac energy metabolism. Partial inhibition of the oxidation of free fatty acids, reducing oxygen free radicals production, reducing calcium overload, exerting mitochondrial protection, antioxidant stress, anti apoptosis and other pharmacological effects, maintaining the production of ATP and the energy metabolism function of ischemic myocardium, thus exerting protective effect on the cardiac muscle, but its effect on autophagy is reported in the literature. Therefore, in this experiment, we used in vitro culture of neonatal rat myocardial cells as research and observation objects, using hypoxia reoxygenation (HR) model in isolated myocardial cells to simulate myocardial ischemia reperfusion injury, and to give different concentrations of trimetazidine intervention. Compare the activity of cardiac myocytes in each group and evaluate the content of LDH in the supernatant to evaluate each group. The damage of cardiac myocytes and the protective effect of trimetazidine on myocardial ischemia reperfusion injury; the detection of autophagic gene Beclin 1 m RNA by RT-PCR to observe the expression of autophagic gene Beclin1 in ischemia reperfusion injury and to explore the effect of trimetazidine on autophagic gene Beclin1, and provide new ideas and basis for clinical use. Methods: 2-3 day healthy Sprague Dawley (SD) rats were born from the animal center of Hebei Medical University to extract and cultivate cardiac myocytes, and divide the cardiomyocytes into 5 groups randomly: 1) normal control group (group A): 10% fetal bovine serum DMEM medium (37, 5%CO2,95% air) culture 26.5h; 2) HR model group (B group): low sugar DMEM medium without serum After 30min culture, 24h was cultured in three gas incubator (92%N2,5%CO2,3%O2), and then 2H simulated ischemic reperfusion injury was cultured in the DMEM medium containing 10% fetal bovine serum (37 C, 5%CO2,95% air); 3) 0.2 mu mol/L triazine preconditioning group (C group): 0.2 micron Trimetazine, low sugar DMEM culture medium culture 30min, established HR model, simulation Ischemic reperfusion (group B); 4) 1 mol/L trimetazidine preconditioning group (group D): 1 mu mol/L Trimetazine and low sugar DMEM culture medium without serum for 30min, establish HR model, simulate ischemia reperfusion (with B group); 5) 5 micron trimetazidine preconditioning group (E group): 5 mu mol/L Trimetazine, low sugar DMEM culture medium culture 3 without serum 3 After 0min, HR model was established to simulate ischemia reperfusion (group B). After the cells were treated, the viability of myocardial cells was detected by thiazolium (MTT) colorimetric assay. The content of lactate dehydrogenase (LDH) was detected and cultured by automatic biochemical analyzer. RT-PCR technique was used to determine the RNA expression of Beclin1 m in each gene. Results: the results of 1 cardiac muscle cell viability: B: B: B The activity of cardiac myocytes in group A was 61.26 + 6.30% (P0.05). The activity of cardiac myocytes in group C, D and E group was 70.34 + 7.17%, 79.33 + 6.90%, 89.43 + 6.02%, respectively higher than that of group B (P0.05), and E group was higher than C group and D group (P0.05), and 2 lactate dehydrogenase assay was 176.96 + 12.70, 157.87 + 13.3. 8 U/L, 126.44 + 12.41U/L, 97.13 + 12.14U/L were significantly higher than that in group A (P0.05). C, D, E group LDH content was lower than B group (P0.05). 3 RT-PCR results: the relative expression of autophagy gene Beclin1 m RNA in B group was significantly higher than that in group A, reaching to group A (P0.05), C group, D group, and E group of cardiomyocyte autophagy gene (2.65 + + 0.20) Group (P0.05), especially in group E, showed that HR could induce the increase of autophagic gene Beclin1 m RNA in cardiac myocytes, while trimetazidine inhibited the expression of Beclin1 m RNA in the autophagy gene, and had a certain concentration dependence. Conclusion: 1 the experiments were made to give low glucose, reoxygenation and reoxygenation conditions to reduce myocardium obviously. Cell vitality, increased LDH content and effectively simulated ischemic reperfusion model.2 Trimetazine can reduce myocardial damage caused by ischemia-reperfusion, and a certain concentration dependent.3 ischemia reperfusion injury can induce the increase of myocardial autophagy Beclin1 m RNA level,.4 Trimetazine can reduce the myocardial finer induced by ischemia-reperfusion. The autophagy gene Beclin1m RNA was expressed in a concentration dependent manner.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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