發(fā)布時(shí)間：2018-09-16 20:55

【摘要】：背景:急性心肌梗死(Acute myocardial infarction)是指冠狀動(dòng)脈在急性的缺血和缺氧的情況下所引起的心肌細(xì)胞的壞死。臨床上患者多有不能緩解的胸骨后疼痛,口服硝酸酯類藥物以及休息后不能或者完全緩解,通常會(huì)出現(xiàn)心電圖的動(dòng)態(tài)改變,血清心肌酶以及心臟損傷標(biāo)志物的升高,通常會(huì)并發(fā)一些惡性心律失常比如:室性心動(dòng)過速、心室顫動(dòng),甚至心源性休克、心臟破裂等,猝死率50%。急性心肌梗死嚴(yán)重危害了人類的生命健康,目前成為全世界最常見的疾病。及早的恢復(fù)冠脈血流,是目前治療的關(guān)鍵,但是在再灌注時(shí)會(huì)出現(xiàn)一系列的損傷作用。預(yù)處理雖可以顯著減輕再灌注損傷,但是缺血的無法預(yù)測在臨床上得不到很好的實(shí)施,從而限制了預(yù)處理的應(yīng)用。2005年,Staat等[1]首次將缺血后處理應(yīng)用于臨床,后處理組與對照組相比減少了36%的心肌梗死的面積。本實(shí)驗(yàn)室已經(jīng)證實(shí)缺血后處理可有效減少缺血再灌注性心律失常、改善心臟的泵功能、減少心肌梗死的面積[2]。同時(shí),機(jī)械后處理的方式可以在再灌注損傷起到保護(hù)作用,但是同時(shí)可能會(huì)存在斑塊的脫落、機(jī)械性血管壁的損傷以及栓塞等風(fēng)險(xiǎn)。藥物后處理(Pharmacology postconditioning)在臨床上可實(shí)施性強(qiáng)。目前藥物后處理藥物有多種,其中常見的有硝酸甘油、地爾硫?、腺苷、尼可地爾等。這四種藥物分別通過不同的機(jī)制對心肌缺血再灌注引起的損傷起到了相應(yīng)的保護(hù)作用,但既往對于這四種藥物之間的治療效果的差異研究甚少,本文旨在研究這四種藥物后處理給藥的方式在離體大鼠心肌缺血再灌注損傷的治療效果的差異,以及探討線粒體ALDH2是否參與其作用機(jī)制的過程,為臨床上單獨(dú)以及聯(lián)合用藥提供一定的理論基礎(chǔ)。目的:1.觀察硝酸甘油、地爾硫?、腺苷以及尼可地爾四種藥物后處理對Wistar離體大鼠心肌缺血/再灌注后左心室心功能(LVDP、±dp/dtmax)、RA、心肌梗死面積的影響,并進(jìn)行藥物之間的對比。2.研究四種藥物后處理對離體大鼠心肌缺血/再灌注過程中心室肌線粒體ALDH2表達(dá)的影響,探討ALDH2是否參與藥物作用機(jī)制的過程。方法:1.藥物后處理對離體大鼠心肌缺血/再灌注損傷影響的對比的研究:將硝酸甘油(10-8mol/L)、地爾硫?(5μmo/L)、尼可地爾(200μmo/L)以及腺苷(100μmo/L),加入到Krebs-Henseleit(K-H)模擬液中,充分震蕩攪勻后備用。將66只品種為Wistar大鼠(雄性)隨機(jī)分為六組:正常組(N組)、缺血再灌注組(I-R組)、硝酸甘油+缺血再灌注組、地爾硫?+缺血再灌注組、尼可地爾+缺血再灌注組、腺苷+缺血再灌注組(各組n=11只)。應(yīng)用Langendorff離體心臟灌流實(shí)驗(yàn)系統(tǒng)以及BL-420S生物機(jī)能實(shí)驗(yàn)系統(tǒng)。正常組(N組)持續(xù)灌流正常液150min;I-R組正常液穩(wěn)定灌流30min后,結(jié)扎前降支30min,繼以正常液再灌注90min;藥物后處理組分別在再灌注即刻給予硝酸甘油、地爾硫?、尼可地爾)以及腺苷再灌注15min,繼以正常液體灌流75min。用3-0號(hào)醫(yī)用縫線的3/8彎針在左心耳根部下2mm處穿過心肌表層,在肺動(dòng)脈圓錐旁出針[3]勾繞冠狀動(dòng)脈前降支,進(jìn)針深度0.5-1mm,寬度為2-3mm[4]。待心臟穩(wěn)定灌流30 min后,實(shí)驗(yàn)組套一球囊并結(jié)扎前降支與球囊,打一活結(jié),球囊內(nèi)加壓至6KPa。此時(shí)會(huì)看到左室心臟表面開始失去原有的紅潤光澤而顯灰白,左心室收縮活動(dòng)減弱以至左心室內(nèi)壓力減半,通過這些現(xiàn)象初步判斷結(jié)扎位置準(zhǔn)確[5],心肌梗死模型建立。結(jié)扎30min后迅速松開球囊,打開結(jié)扎線,再灌注1.5h。最后將藥物后處理組與單純再灌注組大鼠的前降支重新結(jié)扎,2ml濃度1%Evans-blue從主動(dòng)脈的根部逆行灌注心臟,用PBS液體沖洗后迅速置于1%TTC溶液中再染色,區(qū)分正常區(qū)(深藍(lán)色)、缺血未梗死區(qū)(紅色)及梗死區(qū)域(灰白色)。TTC染色后的心臟組織不能用于后續(xù)的分子生物學(xué)研究[6],組織標(biāo)本分開留取。記錄并分析各組左心室功能:左心室發(fā)展壓(LVDP)、左室內(nèi)壓力最大上升/下降速率(±dp/dtmax)、再灌注心律失常(RA)、心肌梗死面積等參數(shù)。2.藥物后處理對離體大鼠心肌缺血再灌注損傷心室肌線粒體ALDH2表達(dá)的影響:動(dòng)物實(shí)驗(yàn)分組同第一部分,本部分采用第一部分離體灌流結(jié)束后留取的左心室心肌組織。其中Real time RT-PCR所用的組織在液氮中保存,Western blot方法所用的組織在-80℃冰箱中保存。實(shí)時(shí)熒光定量逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(Realtime RT-PCR)檢測線粒體ALDH2基因水平的表達(dá)。Western Blot檢測線粒體ALDH2蛋白表達(dá)。結(jié)果:1.1I-R組再灌注30 min及45 min時(shí)LVDP均低于各藥物后處理組(均P0.05)。在藥物后處理組,再灌注30 min地爾硫?組LVDP[(92.68±5.09)mm Hg比(84.26±3.02)mm Hg比(83.35±2.88)mm Hg]、尼可地爾組[(88.95±1.75)mm Hg比(84.26±3.02)mm Hg比(83.35±2.88)mm Hg]與再灌注45 min地爾硫?組LVDP[(90.39±4.29)mm Hg比(82.09±4.24)mm Hg比(80.98±3.89)mm Hg]、尼可地爾組[(86.13±2.38)mm Hg比(82.09±4.24)mm Hg比(80.98±3.89)mm Hg]均高于硝酸甘油組與腺苷組,且地爾硫?組高于尼可地爾組,差異均有統(tǒng)計(jì)學(xué)意義(均P0.05),但硝酸甘油組與腺苷組比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。1.2 I-R組再灌注30 min及45 min的±dp/dtmax均低于各藥物后處理組(均P0.05)。地爾硫?組、尼可地爾組再灌注30 min及45 min的±dp/dtmax均高于硝酸甘油組、腺苷組,且地爾硫?組高于尼可地爾組,差異均有統(tǒng)計(jì)學(xué)意義(均P0.05)。但硝酸甘油組與腺苷組比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。2.RA評分比較:I-R組評分[5(3,6),57.36]均明顯高于各個(gè)藥物后處理組:尼可地爾組[1(1,3),22.05],地爾硫?組[3(1,4),34.77],硝酸甘油組[4(1,4),45.41],腺苷組[2(1,3),23.14],差異均有統(tǒng)計(jì)學(xué)意義(均P0.05)。尼可地爾組RA評分最低,但尼可地爾組與腺苷組差異無統(tǒng)計(jì)學(xué)意義(P=0.771)。3.心肌梗死面積比較:硝酸甘油組(27.04±2.45)%、地爾硫?組(17.01±1.13)%、腺苷組(47.97±1.22)%以及尼可地爾組(34.95±1.25)%均小于I-R組(55.51±1.43)%,差異均有統(tǒng)計(jì)學(xué)意義(均P0.01)。各藥物后處理組組間比較差異均有統(tǒng)計(jì)學(xué)意義(均P0.01),且地爾硫?組梗死面積最小。4.基因與蛋白表達(dá)的結(jié)果:在藥物硝酸甘油、尼可地爾后處理組線粒體ALDH2m RNA表達(dá)分別為0.0657±0.0050、0.0587±0.0004,線粒體ALDH2 m RNA表達(dá)量分別為N組的0.7463±0.0726、0.8339±0.0808倍,與I-R組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05,P0.05)。硝酸甘油與尼可地爾后處理線粒體ALDH2 m RNA表達(dá)差異無統(tǒng)計(jì)學(xué)意義(P0.05)。N組線粒體ALDH2蛋白處于高表達(dá)狀態(tài),I-R組蛋白表達(dá)降低。其中,腺苷、地爾硫?蛋白處于低表達(dá)狀態(tài),硝酸甘油以及尼可地爾蛋白較I-R組相比表達(dá)增加(P0.05)。結(jié)論:1.在心肌缺血再灌注時(shí),硝酸甘油、地爾硫?、腺苷、尼可地爾四種藥物均可改善缺血再灌注后的心臟泵功能、減少再灌注心律失常評分、縮小心肌梗死面積,從而減少心肌缺血再灌注損傷,對心肌起到保護(hù)作用。地爾硫?改善心臟泵功能能力更強(qiáng);在后處理RA方面,尼可地爾以及腺苷效果更好;在減少心肌梗死面積方面,地爾硫?更優(yōu)。本實(shí)驗(yàn)為臨床單獨(dú)以及聯(lián)合用藥方面提供一定的實(shí)驗(yàn)理論基礎(chǔ)。2.腺苷、地爾硫?后處理不能通過上調(diào)線粒體ALDH2相關(guān)機(jī)制對缺血再灌注損傷的心肌起到保護(hù)作用。硝酸甘油、尼可地爾藥物后處理可能通過上調(diào)線粒體ALDH2相關(guān)機(jī)制對缺血再灌注損傷的心肌起到保護(hù)作用。ALDH2可能是硝酸甘油以及尼可地爾發(fā)揮缺血再灌注損傷保護(hù)作用的機(jī)制之一。
[Abstract]:BACKGROUND: Acute myocardial infarction (AMI) refers to the necrosis of myocardial cells caused by acute ischemia and hypoxia of the coronary artery. Acute myocardial infarction (AMI) seriously endangers human life and health and is now the most common disease in the world. Pulse blood flow is the key to current treatment, but a series of injuries occur during reperfusion. Pretreatment can significantly reduce reperfusion injury, but the unpredictability of ischemia can not be well implemented in clinical practice, thus limiting the application of preconditioning. In 2005, Staat et al. [1] first applied ischemic postconditioning to clinical, posterior. Compared with the control group, the area of myocardial infarction was reduced by 36%. Our laboratory has proved that ischemic postconditioning can effectively reduce ischemia-reperfusion arrhythmia, improve cardiac pump function, and reduce the area of myocardial infarction [2]. Pharmacological postconditioning is clinically practicable. There are a variety of post-treatment drugs, including nitroglycerin, diltiazem, adenosine, nicorandil, etc. These four drugs are used to treat myocardium through different mechanisms. Ischemia-reperfusion injury plays a corresponding protective role, but there is little research on the difference of therapeutic effect between these four drugs. This paper aims to study the difference of therapeutic effect of these four drugs on myocardial ischemia-reperfusion injury in isolated rats by post-treatment, and to explore whether mitochondrial ALDH2 participates in it. Objective: 1. To observe the effects of nitroglycerin, diltiazem, adenosine and nicorandil on left ventricular function (LVDP, +dp/dtmax), RA and myocardial infarction area after myocardial ischemia/reperfusion in Wistar rats. To study the effects of four kinds of drug postconditioning on the expression of ALDH2 in ventricular myocardial mitochondria during myocardial ischemia/reperfusion in isolated rats, and to explore whether ALDH2 participates in the mechanism of drug action. Methods: 1. The comparative study of the effects of drug postconditioning on myocardial ischemia/reperfusion injury in isolated rats: Nitroglycerin (10-8 mol/L) Sixty-six Wistar rats (male) were randomly divided into six groups: normal group (N group), ischemia-reperfusion group (I-R group), nitroglycerin + ischemia-reperfusion group, diltiazem + ischemia-reperfusion group, diltiazem + ischemia-reperfusion group. Nicorandil + ischemia-reperfusion group, adenosine + ischemia-reperfusion group (n = 11 rats in each group). Langendorff isolated cardiac perfusion system and BL-420S biological function experimental system were used. Normal group (N group) was perfused for 150 minutes; normal fluid in I-R group was perfused steadily for 30 minutes, the anterior descending branch was ligated for 30 minutes, followed by normal fluid reperfusion for 90 minutes. In the treatment group, nitroglycerin, diltiazem and nicorandil were given immediately after reperfusion for 15 minutes and adenosine for 75 minutes. The needle was inserted into the left atrial appendage 2 mm below the root of the left atrial appendage with a 3/8 curved needle of medical suture No. 3-0. 2-3 mm [4]. After 30 minutes of stable cardiac perfusion, the experimental group set up a balloon and ligated the anterior descending branch and balloon, made a knot, and the balloon was pressurized to 6KPa. At this time, the left ventricular surface began to lose its original red luster and grey, left ventricular systolic activity weakened so that the pressure in the left ventricle halved. Through these phenomena, the ligation was preliminarily judged. Myocardial infarction model was established. After 30 minutes of ligation, the balloon was loosened, the ligation line was opened, and the anterior descending branch was ligated again in the drug treatment group and the reperfusion group for 1.5 hours. The TTC stained cardiac tissue could not be used for follow-up molecular biology studies [6], and the tissue samples were taken separately. Left ventricular function: left ventricular development pressure (LVDP), maximum increase/decrease rate of left ventricular pressure (+dp/dtmax), were recorded and analyzed. Reperfusion arrhythmias (RA), myocardial infarction area and other parameters. 2. Effects of drug postconditioning on the expression of ALDH2 in isolated rat ventricular myocardium after myocardial ischemia-reperfusion injury: Animal experimental group was the same as the first part, this part used the left ventricular myocardial tissue after the first part of perfusion in vitro. Tissues were preserved in liquid nitrogen, and the tissues used in Western blot were stored in a refrigerator at - 80 C. The expression of mitochondrial ALDH2 gene was detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (Realtime RT-PCR). The expression of mitochondrial ALDH2 protein was detected by Western Blot. Results: LVDP in 1.1I-R group was lower than that after reperfusion for 30 min and 45 min. The treatment group (all P 0.05). In the drug treatment group, LVDP [(92.68 [(92.68 [(5.09) mm Hg ratio (84.26 [(92.68 [(5.09) mm Hg ratio (84.26 [(92.68 [(3.02) mm Hg ratio (84.26 [(3.02) mm Hg ratio (83.35 [2.88) mm Hg ratio (83.35 [2.88) mm Hg ratio (83.35 [2.88) mm Hg ratio (83.35 [(83.29) mm Hg ratio] Hg ratio (83.39 [2.88) mm Hg ratio (83.35 [2.88) mm Hg ratio (83.35 [2.mm Hg ratio (80.98) The ratio of nicorandil group [(86.13+2.38)mm Hg to (82.09+4.24)mm Hg to (80.98+3.89)mm Hg] was higher than that of nitroglycerin group and adenosine group, and that of diltiazem group was higher than that of nicorandil group (all P 0.05), but there was no significant difference between nitroglycerin group and adenosine group (P 0.05). The (+) DP / dtmax at 30 min and 45 min of reperfusion in nicorandil group were higher than those in nitroglycerin group and adenosine group, and the (+) DP / dtmax in nicorandil group was higher than that in nicorandil group (all P 0.05). However, there was no significant difference between nitroglycerin group and adenosine group. The scores of I-R group [5 (3,6), 57.36] were significantly higher than those of each drug treatment group: nicorandil group [1 (1,3), 22.05], diltiazem group [3 (1,4), 34.77], nitroglycerin group [4 (1,4), 45.41], adenosine group [2 (1,3), 23.14], the difference was statistically significant (all P 0.05). There was no significant difference in myocardial infarction size between glycoside group and I-R group (P = 0.771). 3. Comparison of myocardial infarction size between nitroglycerin group (27.04 (+ 2.45)), diltiazem group (17.01 (+ 1.13)), adenosine group (47.97 (+ 1.22)) and nicorandil group (34.95 (+ 1.25)) was significantly smaller than that of I-R group (55.51 (+ 1.43)). There were significant differences among the post-treatment groups (all P 0.01). The expression of ALDH2 m RNA in mitochondria was 0.0657 (+ 0.0050) and 0.0587 (+ 0.0004) respectively in nitroglycerin and nicorandil treatment groups, and the expression of ALDH2 m RNA in mitochondria was 0.7463 (+ 0.0726) and 0.8339 (+ 0.08088) times that in N group, respectively. There was no significant difference in the expression of mitochondrial ALDH2 m RNA between nitroglycerin and nicorandil post-treatment (P 0.05). The expression of mitochondrial ALDH2 protein was high in N group, but decreased in I-R group. Conclusion: 1. Nitroglycerin, diltiazem, adenosine and nicorandil can improve cardiac pump function, reduce reperfusion arrhythmia score and myocardial infarction area during myocardial ischemia and reperfusion, thereby reducing myocardial ischemia and reperfusion injury and protecting myocardium. Ertiazem? Is more effective in improving cardiac pump function; nicorandil and adenosine are more effective in treating RA; diltiazem? Is better in reducing myocardial infarction area. This study provides a theoretical basis for clinical use of either alone or in combination. 2. Adenosine, diltiazem? Post-processing can not up-regulate mitochondrial ALDH2-related. Nitroglycerin and nicorandil postconditioning may protect the myocardium from ischemia-reperfusion injury by up-regulating the related mechanisms of mitochondrial ALDH2. ALDH2 may be one of the mechanisms of nitroglycerin and nicorandil in protecting the myocardium from ischemia-reperfusion injury.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R542.22

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