本文選題：缺血再灌注損傷 + 自噬��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【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。隨著人們生活水平的提高,冠心病患者人數(shù)逐年上升,急性心肌梗死(acute myocardial infarction,AMI)患者也明顯增多。目前臨床上治療急性心肌梗死主要采用溶栓、經(jīng)皮冠狀動脈介入治療術(shù)(percutaneous coronary intervention,PCI)以及冠狀動脈旁路移植術(shù)(coronary artery bypass grafting,CABG)等手段以開通阻塞血管,挽救瀕死心肌,但隨之而來的缺血再灌注(ischemia reperfusion,IR)損傷卻嚴(yán)重影響治療效果及患者預(yù)后,因此對于缺血再灌注損傷機制的研究越來越受重視。心肌缺血再灌注損傷涉及多種機制,包括氧自由基增多、鈣超載、炎癥反應(yīng)、線粒體損傷、細(xì)胞凋亡等。近年來的研究表明,自噬是一種新的程序性死亡(II型程序性死亡)方式,成為新的研究熱點,并且被認(rèn)為在心肌缺血再灌注損傷中發(fā)揮著重要作用。自噬是一個在進(jìn)化上高度保守的受損或功能障礙的蛋白質(zhì)或細(xì)胞器降解的過程。適度的自噬對機體起保護(hù)作用,過度的自噬則會加劇細(xì)胞死亡。研究表明,自噬在缺血再灌注損傷中表現(xiàn)出“雙刃劍”的作用,缺血期主要起保護(hù)作用,而再灌注期自噬過度激活則表現(xiàn)出損傷作用,可導(dǎo)致細(xì)胞死亡。自噬的發(fā)生非常復(fù)雜,其中自噬體的形成是自噬發(fā)生的關(guān)鍵環(huán)節(jié)。自噬由大多數(shù)自噬基因(autophagy-related gene,Atg)進(jìn)行調(diào)控,其中Beclin1基因是酵母Atg6的同系物,是形成自噬體的必需分子,可介導(dǎo)自噬相關(guān)基因定位于自噬體膜,并與多種蛋白反應(yīng)調(diào)控自噬體的形成與成熟。因此抑制Beclin1的表達(dá)可有效阻斷自噬的發(fā)生。近年來心肌能量代謝藥物通過聯(lián)合常規(guī)改善血流藥物(硝酸酯類、β-受體阻滯劑、鈣拮抗劑等)治療心肌缺血備受關(guān)注,隨著心肌能量代謝研究的不斷深入,曲美他嗪優(yōu)化心肌代謝的作用機制成為新的研究熱點。曲美他嗪(trimetazidine,TMZ)作為調(diào)節(jié)心肌能量代謝的藥物,通過部分抑制游離脂肪酸的氧化,減少氧自由基產(chǎn)生,減輕鈣超載,發(fā)揮線粒體保護(hù)、抗氧化應(yīng)激、抗細(xì)胞凋亡等多方面的藥理作用,維持ATP的產(chǎn)生及缺血心肌細(xì)胞的能量代謝功能,從而發(fā)揮對心肌的保護(hù)作用。但其對自噬的影響目前文獻(xiàn)報道較少。因此,本實驗以體外培養(yǎng)乳鼠心肌細(xì)胞為研究及觀察對象,利用離體心肌細(xì)胞缺氧復(fù)氧(hypoxia reoxygenation,HR)模型模擬心肌缺血再灌注損傷,并給予不同濃度的曲美他嗪干預(yù)。比較各組心肌細(xì)胞活力、培養(yǎng)上清液LDH含量以評價各組心肌細(xì)胞的受損情況及曲美他嗪對心肌缺血再灌注損傷的保護(hù)作用;采用RT-PCR檢測自噬基因Beclin 1 m RNA水平,以觀察自噬基因Beclin1在缺血再灌注損傷的表達(dá)情況及探討曲美他嗪對自噬基因Beclin1的影響,為臨床用藥提供新思路及依據(jù)。方法:出生2-3天的健康Sprague Dawley(SD)乳鼠,來自河北醫(yī)科大學(xué)動物中心,提取并培養(yǎng)心肌細(xì)胞,并將心肌細(xì)胞隨機分為5組:1)正常對照組(A組):含10%胎牛血清DMEM培養(yǎng)基(37℃、5%CO2、95%空氣)培養(yǎng)26.5h;2)HR模型組(B組):不含血清的低糖DMEM培養(yǎng)基培養(yǎng)30min后,三氣培養(yǎng)箱(92%N2、5%CO2、3%O2)中培養(yǎng)24h,然后在含10%胎牛血清DMEM培養(yǎng)基(37℃、5%CO2、95%空氣)培養(yǎng)2h模擬缺血再灌注損傷;3)0.2μmol/L曲美他嗪預(yù)處理組(C組):含0.2μmol/L曲美他嗪,不含血清的低糖DMEM培養(yǎng)基培養(yǎng)30min后,建立HR模型,模擬缺血再灌注(同B組);4)1.0μmol/L曲美他嗪預(yù)處理組(D組):含1.0μmol/L曲美他嗪,不含血清的低糖DMEM培養(yǎng)基培養(yǎng)30min后,建立HR模型,模擬缺血再灌注(同B組);5)5.0μmol/L曲美他嗪預(yù)處理組(E組):含5.0μmol/L曲美他嗪,不含血清的低糖DMEM培養(yǎng)基培養(yǎng)30min后,建立HR模型,模擬缺血再灌注(同B組);各組細(xì)胞處理后,應(yīng)用噻唑藍(lán)(MTT)比色法檢測心肌細(xì)胞活力、全自動生化分析儀檢測培養(yǎng)清乳酸脫氫酶(LDH)含量、采用RT-PCR技術(shù)測定各組自噬基因Beclin1 m RNA表達(dá)水平。結(jié)果:1心肌細(xì)胞活力結(jié)果:B組心肌細(xì)胞活力為A組的61.26±6.30%(P0.05),C組、D組、E組心肌細(xì)胞活力分別為A組的70.34±7.17%、79.33±6.90%、89.43±6.02%,均明顯高于B組(P0.05),其中E組高于C組、D組(P0.05);2乳酸脫氫酶(LDH)測定結(jié)果:B組、C組、D組、E組LDH含量分別為176.96±12.70 U/L、157.87±13.38 U/L、126.44±12.41U/L、97.13±12.14U/L均明顯高于A組的65.18±16.99U/L(P0.05),C組、D組、E組LDH含量均低于B組(P0.05),E組低于C組、D組(P0.05),與心肌細(xì)胞活力結(jié)果共同說明通過HR建立的離體IR模型是有效的,HR造成離體心肌細(xì)胞損傷,而曲美他嗪能減輕HR誘導(dǎo)的心肌細(xì)胞損傷,并具有一定的濃度依賴性;3 RT-PCR結(jié)果:B組心肌細(xì)胞自噬基因Beclin1 m RNA相對表達(dá)量明顯高于A組,達(dá)A組的2.99±0.19倍(P0.05),C組、D組、E組心肌細(xì)胞自噬基因Beclin1 m RNA相對表達(dá)量分別為(2.65±0.24、2.19±0.17、1.64±0.20)均低于B組(P0.05),尤以E組明顯,說明HR可誘導(dǎo)心肌細(xì)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的表達(dá),并具有一定的濃度依賴性。結(jié)論:1本實驗通過培養(yǎng)乳鼠心肌細(xì)胞給予低糖、缺氧后復(fù)糖、復(fù)氧條件,能明顯降低心肌細(xì)胞活力,增加LDH含量,有效模擬缺血再灌注模型。2曲美他嗪能減輕缺血再灌注引起的心肌細(xì)胞損傷,并具有一定的濃度依賴性。3缺血再灌注損傷可誘導(dǎo)心肌細(xì)胞自噬基因Beclin1 m RNA水平增加。4曲美他嗪可減少缺血再灌注誘導(dǎo)的心肌細(xì)胞自噬基因Beclin1m RNA的表達(dá),并具有一定的濃度依賴性。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R54

【參考文獻(xiàn)】

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1 李Z，

本文編號：1907070