MicroRNA-155对急性心肌梗死后心室重构的影响及机制探讨

发布时间：2018-04-24 06:50

本文选题：心肌梗死 + 心室重构　；参考：《武汉大学》2016年博士论文

【摘要】：背景：急性心肌梗死(acute myocardial infarction, AMI)是指因冠状动脉斑块破裂或痉挛导致冠脉血管急性、持续性闭塞而引起心肌组织缺血缺氧导致心肌坏死。AMI后,在免疫炎症因子、神经体液紊乱、心肌缺血和心脏负荷异常等因素作用下导致心室重构,包括心肌细胞及细胞间质结构、功能和表型等异常改变。如何预防和治疗AMI后心室重构成为当今心血管病学研究的热点和主要挑战。microRNAs(miRNAs)是一类约含22个核苷酸的非编码小RNA分子,通过与靶基因mRNA的3'-UTR区结合抑制其翻译或致其降解,在转录后水平上影响基因表达,发挥重要生物功能。多种炎症疾病中发现microRNA-155(miR-155)表达上调,如类风湿性关节炎和动脉粥样硬化等。AMI患者血清中miR-155的表达显著上调,并与患者1年内的死亡风险密切相关。但是miR-155在AMI后心室重构中的作用及其机制,特别是对心脏成纤维细胞的作用仍不清楚。本研究利用miR-155过表达和miR-155敲除小鼠,通过结扎小鼠冠状动脉前降支(LAD)建立AMI模型,研究miR-155对AMI后心脏功能和心室重构的影响,并探讨其潜在的作用机制。第一部分：miR-155对小鼠AMI后心脏功能及心室重构的影响目的：探讨miR-155对小鼠AMI后心脏功能及心室重构的影响。方法：利用miR-155基因敲除(miR-155-/-)、基因过表达(miR-155+／+)及野生型(Wild Type, WT)小鼠,通过结扎LAD近端建立AMI模型,假手术组(sham)小鼠穿针不结扎。在术后第1,7和14天通过心脏超声观察小鼠的左心室结构及功能改变。术后14天,通过TTC染色和Masson染色检测小鼠心肌梗死面积、左室扩张指数以及疤痕的厚度。免疫组化检测小鼠心肌脑钠肽(BNP)和心肌肌钙蛋白I (cTnI)的表达变化。结果： (1)与sham组相比,心梗后第1,7和14天小鼠心脏功能各项指标明显受损。miR-155-/-组小鼠心梗后心功能明显优于WT组和miR-155+/+组小鼠。(2)TTC染色发现miR-1554-小鼠的梗死面积明显小于WT和miR-155+/+小鼠。miR-155+/+小鼠的梗死面积明显大于WT和miR-155-/-小鼠。(3) Masson染色发现miR-155-/-小鼠的相对瘢痕厚度明显低于WT和miR-155+-小鼠。miR-155-/-小鼠的左室扩张指数明显低于WT和miR-155+/+小鼠。 (4)miR-155-/-小鼠心肌中BNP和cTnI的表达显著低于WT和miR-155+／+小鼠。结论：miR-155加重小鼠AMI后心脏功能的恶化和心肌损伤,促进了心梗后心室不良重塑过程。第二部分miR-155对小鼠心脏成纤维细胞胶原蛋白合成、凋亡、增殖和表型转化的影响目的：心肌纤维化是AMI后心室重构的重要过程。心脏成纤维细胞在心肌纤维化过程中起主导作用。本部分旨在探讨miR-155对小鼠心脏成纤维细胞胶原蛋白合成、凋亡、增殖和表型转化等生物行为的影响。方法：分离WT、miR-155-/-和miR-155+/+小鼠AMI 14天后的心脏成纤维细胞和心肌细胞,使用RT-PCR法检测miR-155和肿瘤p53蛋白诱导核蛋白1(TP53INP1)的表达变化。转化生长因子-β1 (TGF-β1)与乳鼠心脏成纤维细胞共培养24h、48h、72h后,使用RT-PCR法检测成纤维细胞中miR-155和Ⅰ/Ⅲ型胶原蛋白mRNA及caspase-3的表达变化。利用Western blotting检测成纤维细胞中cleaved caspase-3表达变化。利用MTT法检测TGF-β1刺激心脏成纤维细胞后增殖情况。利用免疫荧光法检测平滑肌肌动蛋白-α (α-SMA)的表达。结果： (1)与sham组比较,AMI组WT小鼠心脏成纤维细胞中miR-155的表达显著增加,而TP53INP1表达显著降低,而心肌细胞中miR-155和TP53INP1均无明显变化。心肌细胞和心脏成纤维细胞中TP53INP1的表达在miR-155+/+小鼠AMI后显著下调,而在miR-155-/-小鼠显著上调。(2) TGF-β1刺激WT小鼠心脏成纤维细胞后其miR-155表达水平显著上调,且呈时间依赖性,Ⅰ/Ⅲ型胶原蛋白表达水平显著上调,其表达在miR-155+/+组更显著,而miR-155敲除后能够抑制TGF-β1介导的collagen Ⅰ/Ⅲ上调。(3)在WT小鼠心脏成纤维细胞中,TGF-β1刺激使caspase-3的mRNA表达水平及其剪切体蛋白(cleaved caspase-3)水平显著下调。miR-155+/+组caspase-3的mRNA表达水平明显低于野生型组,而miR-155基因敲除能够逆转TGF-β1介导的caspase3表达下调。 (4)与WT相比,miR-155过表达显著促进心脏成纤维细胞的增殖,而敲除miR-155后则抑制其增殖。 (5)miR-155+/+组心脏成纤维细胞α-SMA的表达水平显著升高,而miR-155-/-组的心脏成纤维细胞α-SMA的表达水平则显著降低。结论：小鼠AMI后心脏成纤维细胞miR-155表达显著增加,促进成纤维细胞的增殖和Ⅰ/Ⅲ型胶原蛋白的合成,抑制成纤维细胞凋亡,并诱导其向肌成纤维细胞转化,其机制可能与抑制TP53INP1表达有关。第三部分：miR-155靶向调节TP53INP1影响心脏成纤维细胞胶原蛋白合成、凋亡、增殖和表型转化目的：明确miR-155是否通过靶向调节TP53INP1影响心脏成纤维细胞胶原蛋白合成、凋亡、增殖和表型转化方法：通过生物信息学预测miR-155的靶基因并用荧光素酶报告基因检测验证。miR-155模拟物或抑制剂转染乳鼠心脏成纤维细胞,检测miR-155对心脏成纤维细胞中TP53INP1表达的影响。合成靶基因TP53INP1的小片段干扰核糖核酸(siRNA),转染野生型或miR-1554-小鼠的心脏成纤维细胞,检测各组心脏成纤维细胞中Ⅰ/Ⅲ型胶原蛋白和caspase-3 mRNA及蛋白表达变化,MTT法检测心脏成纤维细胞增殖。免疫荧光检测各组a-SMA的表达变化。结果：(1)生物信息学预测TP53INP1是miR-155的靶基因,荧光素酶报告基因实验显示miR-155能与TP53INP1基因3'-UTR结合。心脏成纤维细胞中miR-155模拟物显著抑制TP53INP1的mRNA和蛋白表达水平,而miR-155抑制剂显著增加TP53INP1的mRNA和蛋白表达水平。(2) TP53INP1沉默后成纤维细胞合成Ⅰ/Ⅲ型胶原蛋白显著增加,caspase-3的表达水平下降。(3)在野生型和miR-1554-的心脏成纤维细胞中,与阴性对照组相比,TP53INP1干扰后显著促进心脏肌成纤维细胞的增殖,心脏成纤维细胞α-SMA的表达水平显著增加。结论：miR-155通过靶向调控TP53INP1的表达,促进心脏成纤维细胞的增殖、胶原合成和表型转化,并抑制成纤维细胞凋亡,与心梗后心肌纤维化有关。
[Abstract]:Background: acute myocardial infarction (acute myocardial infarction, AMI) refers to the causes of acute myocardial infarction caused by coronary artery plaque rupture or spasm resulting in myocardial ischemia and hypoxia caused by myocardial ischemia and hypoxia caused by persistent occlusion of the coronary artery, and it leads to immune inflammatory factors, neurohumoral disorders, myocardial ischemia, and abnormal cardiac load. Ventricular remodeling, including abnormal changes in cardiac myocytes and interstitial structures, functions and phenotypes. How to prevent and treat ventricular remodeling after AMI has become a hot and major challenge in current cardiovascular disease research.MicroRNAs (miRNAs) is a class of non coded small RNA molecules containing approximately 22 nucleotides, binding to the 3'-UTR region of the target gene mRNA. MicroRNA-155 (miR-155) expression up-regulated in a variety of inflammatory diseases, such as rheumatoid arthritis and atherosclerosis, the expression of miR-155 in the serum of.AMI patients, such as rheumatoid arthritis and atherosclerosis, is up significantly up, and is closely related to the risk of death within 1 years of the patient. However, the role of miR-155 in ventricular remodeling after AMI and its mechanism, especially for cardiac fibroblasts, is still unclear. This study used miR-155 overexpression and miR-155 knockout mice to establish AMI model by ligating the anterior descending branch of coronary artery (LAD) in mice to study the effect of miR-155 on cardiac function and ventricular remodeling after AMI, and to explore the effect of miR-155 on cardiac function and ventricular remodeling. Potential mechanism of action. Part 1: the effect of miR-155 on cardiac function and ventricular remodeling in mice after AMI: To explore the effect of miR-155 on cardiac function and ventricular remodeling in mice after AMI. Methods: using miR-155 gene knockout (miR-155-/-), gene overexpression (miR-155+ / +) and wild type (Wild Type, WT) mice, by ligation LAD near The AMI model was established at the end of the group, and the sham operation group (sham) was not ligation. The left ventricular structure and function of the mice were observed by echocardiography at 1,7 and 14 days after the operation. The infarct area, the left ventricular dilatation index and the thickness of the scar were detected by TTC staining and Masson staining on the 14 day after operation. Changes in expression of BNP and cardiac troponin I (cTnI). Results: (1) compared with group sham, the cardiac function indexes of mice after 1,7 and 14 days after myocardial infarction were significantly impaired in.MiR-155-/- group after myocardial infarction. (2) TTC staining found that the infarct area of miR-1554- mice was significantly smaller than WT and miR-155+/+. The infarct area of mouse.MiR-155+/+ mice was significantly greater than that of WT and miR-155-/- mice. (3) Masson staining showed that the relative scar thickness of miR-155-/- mice was significantly lower than that of WT and miR-155+- mice, and the left ventricular dilatation index of.MiR-155-/- mice was significantly lower than that of WT and miR-155+/+ mice. (4) the expression of BNP in the miR-155- / - mouse myocardium was significantly lower than that of the miR-155+/+ mice. And miR-155+ / + mice. Conclusion: miR-155 aggravates the deterioration of cardiac function and myocardial injury after AMI in mice, promotes the process of ventricular remodeling after myocardial infarction. Second the effect of part miR-155 on collagen synthesis, apoptosis, proliferation and phenotypic transformation of cardiac fibroblasts in mice: myocardial fibrosis is an important part of ventricular remodeling after AMI Cardiac fibroblasts play a leading role in the process of myocardial fibrosis. This part aims to explore the effects of miR-155 on the biological behavior of collagen synthesis, apoptosis, proliferation and phenotypic transformation of cardiac fibroblasts in mice. Methods: isolation of cardiac fibroblasts and cardiac myocytes from WT, miR-155-/- and miR-155+/+ mice for 14 days after AMI. The expression of miR-155 and tumor p53 protein induced nucleoprotein 1 (TP53INP1) expression was detected by RT-PCR. TGF - beta 1 (TGF- beta 1) was co cultured with 24h, 48h, and 72h in rat heart fibroblasts. The expression of mRNA and Caspase-3 in fibroblasts was detected by RT-PCR method. The expression of cleaved caspase-3 in fibroblasts was detected. The proliferation of TGF- beta 1 stimulated cardiac fibroblasts was detected by MTT method. The expression of smooth muscle actin - alpha (alpha -SMA) was detected by immunofluorescence. Results: (1) the expression of miR-155 in cardiac fibroblasts of WT mice of AMI group increased significantly compared with sham group, and TP53INP in WT mice of AMI group increased significantly, and TP53INP The expression of 1 was significantly decreased, but there was no significant change in miR-155 and TP53INP1 in cardiac myocytes. The expression of TP53INP1 in cardiac myocytes and cardiac fibroblasts decreased significantly after AMI in miR-155+/+ mice, and in miR-155-/- mice significantly up. (2) TGF- beta 1 stimulated the miR-155 expression level of WT mice after cardiac fibroblasts. Inter dependence, the expression level of type I / III collagen was significantly up-regulated, and its expression was more significant in miR-155+/+ group, while miR-155 knockout could inhibit the up regulation of collagen I / III mediated by TGF- beta 1. (3) in WT mouse cardiac fibroblasts, TGF- beta 1 stimulated the level of Caspase-3 mRNA expression and the level of cleaved caspase-3 (cleaved caspase-3). The mRNA expression level of Caspase-3 in.MiR-155+/+ group was significantly lower than that in the wild type group, while miR-155 gene knockout could reverse the TGF- beta 1 mediated Caspase3 expression. (4) over expression of miR-155 significantly promoted the proliferation of cardiac fibroblasts compared with WT, and the proliferation was inhibited after miR-155. (5) cardiac fibroblasts in the miR-155+/+ group The expression level of alpha -SMA increased significantly, while the expression level of alpha -SMA in cardiac fibroblasts in group miR-155-/- decreased significantly. Conclusion: the expression of miR-155 in cardiac fibroblasts in mice after AMI was significantly increased, promoting the proliferation of fibroblasts and the synthesis of type I / III collagen, inhibiting the apoptosis of fibroblasts and inducing it to myofibroblast. Cell transformation, its mechanism may be related to the inhibition of TP53INP1 expression. Third part: miR-155 targeting regulation of TP53INP1 affects the collagen synthesis, apoptosis, proliferation and phenotypic transformation of cardiac fibroblasts: to determine whether miR-155 affects collagen synthesis, apoptosis, proliferation and phenotypic transformation of cardiac fibroblasts through targeting regulation of TP53INP1 Methods: using bioinformatics to predict the target gene of miR-155 and using luciferase reporter gene detection to verify the effect of miR-155 on the expression of TP53INP1 in cardiac fibroblasts, the effect of the.MiR-155 analog or inhibitor on the expression of TP53INP1 in the cardiac fibroblasts. The small fragment interfering ribonucleic acid (siRNA) of the target gene TP53INP1, and the transfection field The expression of type I / III collagen and caspase-3 mRNA and protein in cardiac fibroblasts were detected in the cardiac fibroblasts of miR-1554- mice. The proliferation of cardiac fibroblasts was detected by MTT method. The expression of a-SMA in each group was detected by immunofluorescence. Results: (1) the prediction of TP53INP1 was the target gene of miR-155, and the fluorescence of TP53INP1 was the target gene of miR-155. MiR-155 can bind to the TP53INP1 gene 3'-UTR. MiR-155 mimics in cardiac fibroblasts significantly inhibit the mRNA and protein expression level of TP53INP1, while miR-155 inhibitors significantly increase the mRNA and protein expression level of TP53INP1. (2) TP53INP1 after silencing of TP53INP1, the synthesis of type I / III collagen in fibroblasts The expression level of Caspase-3 decreased. (3) in the wild and miR-1554- cardiac fibroblasts, compared with the negative control group, the TP53INP1 interference significantly promoted the proliferation of cardiac myofibroblast and the expression level of the alpha -SMA in the cardiac fibroblasts increased significantly. Conclusion: miR-155 promotes the expression of TP53INP1 by targeting the target and promotes the expression of miR-155. The proliferation, collagen synthesis and phenotypic transformation of cardiac fibroblasts, and the inhibition of apoptosis of fibroblasts, are related to myocardial fibrosis after myocardial infarction.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.22

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