miR-208a在心肌缺血再灌注损伤中作用及相关机制的研究

发布时间：2018-05-21 08:29

本文选题：心肌缺血再灌注损伤 + microNAs　；参考：《昆明医科大学》2016年博士论文

【摘要】：研究背景目前,开胸手术已被广泛用于治疗各种类型的心脏疾病,体外循环是开胸心脏手术中至关重要的手段。尽管如此,一些研究揭示了这种手术本身能够引起人体多种器官的损伤,其中多数伴随着组织器官的缺血再灌注损伤。因此,研究缺血再灌注损伤的机制,尝试规避或减弱这一临床问题将为患者带来益处。miRNAs是短片段单链非编码RNAs,它能够靶向mRNAs进行转录后调控。不断积累的研究表明了miRNAs在缺血再灌注损伤的发展过程中扮演重要角色,其可能成为治疗心肌缺血再灌注损伤的潜在靶点。目的：本研究通过尝试了解体外循环的心脏手术过程中一些与心肌有关的miRNAs变化,并对miR-208a这一关键miRNA进行深入研究,利用大鼠缺血再灌注模型检测miR-208a在缺血再灌注损伤中的作用。以期为缺血再灌注的早期诊断、防治提供理论依据,为未来的分子治疗提供潜在靶点。方法：1, qRT-PCR检测15例风心病患者体外循环的开胸手术过程中miR-1/21/208a/499分别在四个时间点的表达水平(术前、主动脉阻断45 mmin后、再灌注60mmin后、术后24h)；检测传统生理生化指标肌酸激酶同工酶(CK-MB)和肌钙蛋白1(cTnI)的水平；通过统计学分析检测它们之间的相关性。2,构建大鼠缺血再灌注模型,将Wistar大鼠随机分为假手术组(Sham)和缺血再灌注组(I/R),Sham组大鼠仅开胸,I/R组开胸结扎冠状动脉左前降支45min后再灌注120min。荧光定量PCR检测miR-208a的表达变化；Evans蓝染色和氯化三苯基四氮唑(triphenyl tetrazolium chloride, TTC)染色来检测心肌梗死情况;HE染色检测心肌组织形态结构变化；末端脱氧核苷酸转移酶介导的末端标记技术(TUNEL)检测各组心肌细胞凋亡情况；Western blot检测心肌细胞凋亡标志物Caspase-3的变化。3,采用antagomir调控miRNA的表达,检测是否能够减缓缺血再灌注损伤：构建大鼠缺血再灌注模型,将大鼠随机分为缺血再灌注组(I/R)、control antagomir注射后再行缺血再灌注组(I/R+anta-control), miR-208a antagomir注射后再行缺血再灌注组(I/R+anta-miR-208a). I/R组开胸结扎前降支45min后再灌注120min。I/R+anta-control或I/R+anta-miR-208a为缺血再灌注前2天注射2nmol剂量的相应antagomir。荧光定量PCR检测miR-208a的表达变化；Evans蓝染色和TCC检测心肌梗死情况；HE染色检测心肌组织形态结果变化；TUNEL法检测各组心肌细胞凋亡情况；Western blot检测心肌细胞凋亡标志物Caspase-3的变化。荧光定量PCR检测心房利钠因子(atrial natriuretic factor,ANF)和脑钠肽(brain natriuretic peptide, BNP)的表达水平,同时采用LDH检测试剂盒检测各组大鼠血清LDH水平以评估心肌功能。4,通过生物信息学的方法预测miR-208a的潜在靶向mRNA,通过荧光素酶报告实验初步筛选miR-208a的靶基因,并运用流式细胞术检测转染后各组细胞的凋亡情况。结果：1, miR-1/208a/499的表达在体外循环前和再灌注后出现了明显的变化(P 0.05), miR-21在本研究中未观测到变化(P0.05)。miR-1/208a的表达水平在主动脉阻断45min时出现显著变化(P0.05),但当再灌注60min后显著增加(P0.05),并持续至术后24h(P0.05)。miR-1/208a的表达变化模式同传统生理指标CK-MB和cTnl的水平变化相似,而且miR-208a的变化较miR-1迅速。与之相反,miR-499的表达水平在再灌注后持续降低一直到术后24h(P0.05)。而且它的变化同CK-MB和cTnl的水平变化呈负相关。2,通过心电图和染色检测结果表明,大鼠的心脏缺血再灌注损伤模型成功构建。与Sham组相比较,I/R组的大鼠心肌缺血区miR-208a显著升高(P0.05)；细胞凋亡率明显增加(P0.05),凋亡标志物Caspase-3表达水平升高(P0.05),剪切体被激活。这暗示了miR-208a可能与心肌细胞的凋亡存在一定的关联性。3,大鼠转染antagomir后进行缺血再灌注损伤造模,随后进行各项指标的检测。结果发现,I/R+anta-miR-208a组中的miR-208a的表达水平明显低于其余两组(I/R和I/R+anta-control) (P0.05),表达明显受到抑制。TCC检测结果发现I/R+anta-miR-208a组的心肌梗死范围明显少于其余两组(P0.05)；TUNEL检测结果显示I/R+anta-miR-208a组的心肌细胞凋亡率显著降低,Caspase-3表达水平也同样显著降低(P0.05); ANF和BNP以及血清中LDH的水平在anta-miR-208a处理后均显著降低(P0.05),说明心肌功能障碍有所减轻。各个指标在I/R和I/R+anta-control两组中均无显著性差异(P0.05)。4,根据生物信息学分析,我们预测miR-208a与GATA4和QKI5的3'UTR区域有直接的结合位点。进一步对之前的样本进行GATA4和QKI5表达水平检测发现,I/R组的GATA4和QKI5表达水平较Sham组显著降低；与I/R组相比,抑制miR-208a预处理能够升高GATA4和QKI5的表达,二者存在负相关性。双荧光素酶报告基因检测证实了miR-208a同GATA4而非QKI5的靶向关系。结论：1,在体外循环的开胸手术过程中,miR-1/208a/499出现了显著变化,其中miR-208a反应更加迅速。2,在大鼠缺血再灌组损伤模型中,降低miR-208a的表达水平能够明显抑制心肌细胞的凋亡,减缓由于缺血再灌注所导致的心肌功能障碍,具有心脏保护作用。3, miR-208a的这种作用可能通过靶向GATA4或影响QK15相关信号通路来实现。
[Abstract]:At present, thoracotomy has been widely used in the treatment of various types of heart diseases. Cardiopulmonary bypass is a vital means in open heart surgery. However, some studies have revealed that the operation itself can cause damage to various organs in the body, most of which are accompanied by ischemia-reperfusion injury in tissues and organs. Studying the mechanism of ischemia-reperfusion injury, trying to avoid or weaken this clinical problem will benefit the patient.MiRNAs is short fragment single strand non coding RNAs, which can target mRNAs after transcriptional regulation. Continuous accumulation of research shows that miRNAs plays an important role in the development of ischemia-reperfusion injury, and it may become a treatment. Objective: To explore the potential targets of myocardial ischemia reperfusion injury. Objective: To investigate the changes of miRNAs related to myocardium during cardiopulmonary bypass in cardiopulmonary bypass, and to study the key miRNA of miR-208a, and to detect the role of miR-208a in ischemia reperfusion injury by rat ischemia-reperfusion model. The early diagnosis of blood reperfusion provides a theoretical basis for prevention and treatment, and provides potential targets for future molecular therapy. Methods: 1, qRT-PCR was used to detect the level of expression of miR-1/21/208a/499 at four time points during open thoracic surgery for cardiopulmonary bypass in 15 patients with rheumatic heart disease (pre operation, 45 mmin of main artery occlusion, reperfusion after 60mmin, postoperative 24h); The levels of the traditional physiological and biochemical indexes of creatine kinase isoenzyme (CK-MB) and troponin 1 (cTnI) were detected. By statistical analysis, the correlation.2 between them was detected and the rat model of ischemia reperfusion was constructed. The rats were randomly divided into the sham operation group (Sham) and the ischemia reperfusion group (I/R). The rats in the Sham group were only open to the chest, and the I/R group opened the chest to ligation coronal. The expression of miR-208a was detected by 120min. fluorescence quantitative PCR reperfusion after the left anterior descending branch of the artery 45min; Evans blue staining and three phenyl tetrazolium chloride (triphenyl tetrazolium chloride, TTC) staining were used to detect myocardial infarction; HE staining was used to detect the changes in the morphological structure of the myocardium; terminal deoxynucleotidyl transferase mediated terminal labeling The technique (TUNEL) was used to detect the apoptosis of cardiac myocytes in each group; Western blot was used to detect the changes of myocardial apoptosis marker Caspase-3,.3, and antagomir to regulate the expression of miRNA, and to detect whether the ischemia reperfusion injury could be slowed down: the rat model of ischemia reperfusion was constructed and the rats were randomly divided into ischemic reperfusion group (I/R), control antagomir Ischemia-reperfusion group (I/R+anta-control) was performed after injection, and ischemia reperfusion group (I/R+anta-miR-208a) was performed after miR-208a antagomir injection. I/R group was injected with 120min.I/R+anta-control or I/R+anta-miR-208a before ligation of thoracic ligation, and 120min.I/R+anta-control or I/R+anta-miR-208a was injected at 2 days before ischemia-reperfusion, and the corresponding antagomir. fluorescence quantitative PCR detection of miR-2 was detected. 08A expression changes; Evans blue staining and TCC detection of myocardial infarction; HE staining to detect changes in myocardial tissue morphology; TUNEL method to detect the apoptosis of cardiac myocytes in each group; Western blot to detect the change of Caspase-3 in cardiac myocyte apoptosis marker. Fluorescence quantitative PCR detection of atrial natriuretic factor (atrial natriuretic factor,) and The expression level of brain natriuretic peptide (BNP) and LDH detection kit were used to detect the serum LDH level in each group to evaluate the myocardial function.4. The potential target mRNA of miR-208a was predicted by bioinformatics. The target gene of miR-208a was screened by the luciferase reporter experiment and the flow cytometry was used to detect the target gene. Results: 1, 1, the expression of miR-1/208a/499 appeared obvious changes before and after cardiopulmonary bypass (P 0.05). The expression level of miR-21 in this study was not observed in this study (P0.05), and the expression level of.MiR-1/208a was significantly changed when the aorta blocked 45min (P0.05), but increased significantly after reperfusion of 60min. In addition (P0.05), the expression pattern of 24h (P0.05).MiR-1/208a was similar to that of the traditional physiological indexes of CK-MB and cTnl, and the change of miR-208a was faster than that of miR-1. The results of electrocardiogram and staining showed that the model of myocardial ischemia reperfusion injury was successfully constructed in rats. Compared with group Sham, the myocardial ischemia area of rats in group I/R increased significantly (P0.05), the apoptosis rate increased significantly (P0.05), the level of Caspase-3 expression of apoptosis marker increased (P0.05), and the shear body was activated in I/R. This suggests that miR-208a may have a certain correlation with the apoptosis of cardiac myocytes, the rat model of ischemia reperfusion injury after transfection of antagomir, and then the detection of various indexes. The results showed that the expression level of miR-208a in the I/R+anta-miR-208a group was significantly lower than that of the remaining two groups (I/R and I/R+anta-control) (P0.05), and the expression of miR-208a in the I/R+anta-miR-208a group was clearly expressed. The results of.TCC detection showed that the infarct range in group I/R+anta-miR-208a was significantly less than that of the other two groups (P0.05); TUNEL detection showed that the apoptosis rate of myocardial cells in I/R+anta-miR-208a group decreased significantly, and the level of Caspase-3 expression decreased significantly (P0.05), ANF and BNP and the level of LDH in serum were in anta-miR-208a. There was a significant decrease (P0.05), indicating a reduction in myocardial dysfunction. Each index had no significant difference in I/R and I/R+anta-control two groups (P0.05).4. According to bioinformatics analysis, we predicted a direct binding site between miR-208a and the 3'UTR region of GATA4 and QKI5. Further GATA4 and QKI5 expressed water for the previous samples. The level of GATA4 and QKI5 expression in the I/R group was significantly lower than that in the Sham group. Compared with the I/R group, the inhibition of miR-208a preconditioning could increase the expression of GATA4 and QKI5, and there was a negative correlation between the two. The double luciferase reporter gene test confirmed the target relationship between miR-208a and GATA4 but not QKI5. Conclusion: 1, open thoracotomy in cardiopulmonary bypass. During the process, there are significant changes in miR-1/208a/499, in which the miR-208a reaction is more rapid.2. In the rat model of ischemia reperfusion injury, the decrease of the expression level of miR-208a can obviously inhibit the apoptosis of cardiac myocytes and slow down the myocardial dysfunction due to ischemia reperfusion, which has the effect of cardiac protection,.3, miR-208a. It may be achieved by targeting GATA4 or affecting QK15 related signaling pathways.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.2

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