羊急性心梗卸负荷模型的miRNA表达谱

发布时间：2018-03-01 20:49

本文关键词： MiRNA 表达谱急性心梗左心室辅助装置(LVAD)　出处：《北京协和医学院》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景及目的心肌细胞因缺乏再生能力,遇到缺血缺氧等多种原因将导致心肌细胞损伤,造成心肌细胞丢失、纤维组织增生,引发心室重构,并逐渐进展成心力衰竭。左心室循环辅助装置(LVAD)已经被证明是治疗心力衰竭的有效手段,使用LVAD进行机械循环支持可以通过对衰竭的左心室卸负荷的方式带来有利的影响。然而,LVAD在急性心肌梗死治疗的应用还需进一步研究论证,LVAD对AMI后期的预后及其机制有待进一步阐明。微小RNA(miRNA,miRNA)是一种21-23个碱基组成的内源性非编码单链小分子RNA,可在转录后水平调控基因的表达。MiRNA参与多种病理生理学过程。目前miRNAs在心肌缺血中的功能与机制研究尚处于起步阶段,尤其是有关心肌缺血早期miRNAs改变的报道较少。也没有在AMI早期采取LVAD干预后,对心肌细胞内部miRNA的分析,及其相关信号通路的研究。本文试图在羊急性心梗模型上建立miRNA表达谱,为LVAD治疗急性心梗提供理论依据。研究方法1、成年小尾寒羊经结扎冠状动脉制作心梗模型,心脏不停跳植入FW-2型轴流泵,分别为对照组和卸负荷组。2、模型建立后,进行心电图检测,同时置入Swan-gaze漂浮导管进行检测记录。3、72小时后,分别取对照组和卸负荷组的心肌组织,分为正常区域,梗死边带区域以及梗死区域,进行组织染色法。4、TUNEL法检测心肌细胞的凋亡情况。5、高通量测序技术建立miRNA表达谱。6、比较对照组和卸负荷组相应区域心肌中差异表达的miRNA。7、PCR对测序结果进行验证。8、验证后对差异表达的miRNA进行功能和信号通路分析。研究结果1、对照组动物实验共进行7只,其中3只造模成功。LVAD组动物实验共有5只,其中3只成功。2、HE染色结果显示与对照组相比,卸负荷组心肌细胞破坏减弱,同时炎症细胞浸润明显减少。Masson染色结果显示卸负荷组胶原化面积减小,心肌细胞保留较多。天狼猩红染色结果显示卸负荷组胶原化明显减轻;对比梗死区域,卸负荷后主要是Ⅲ型胶原,而对照组则主要是Ⅰ型胶原。3、TUNEL结果显示心梗卸负荷后,心肌细胞凋亡明显减弱。4、卸负荷组与对照组相比,心梗区域差异表达的miRNA是oar-miR-19b(上调);梗死边带区域差异表达的miRNA有3个,其中下调的是oar-miR41 1b-5p 和 oar-miR-487a-5p,上调的是 oar-miR-26a。5、PCR验证结果显示 oar-miR-19b(P0.05)和 oar-miR-26a(P0.01)在卸负荷组与对照组中表达有明显差异,与芯片结果相符。6、GO和KEGG分析结果显示oar-miR-19b和oar-miR-26a所涉及到的功能和信号通路均与心血管疾病有密切的关系。结论本实验利用FW-2型轴流泵成功地对急性心梗的羊进行了 3天的循环辅助,证实LVAD可有效减轻心梗程度。成功地建立了急性心梗和LVAD干预的miRNA表达谱,并进行了验证,结果表明该表达谱可用于探索未知的miRNA,并研究它们参与急性心梗反应的调控机理。
[Abstract]:Background and objective Myocardial cells, due to lack of regenerative ability, may lead to myocardial cell damage, myocardial cell loss, fibrous tissue proliferation and ventricular remodeling when they encounter ischemia and hypoxia. Left ventricular circulatory aids (LVAD) have been shown to be effective in the treatment of heart failure. Mechanical circulatory support with LVAD may have a beneficial effect on the left ventricular load relief from failure. However, the application of LVAD in the treatment of acute myocardial infarction needs further study to demonstrate its late prognosis and prognosis in patients with acute myocardial infarction. The mechanism of microRNAs miRNAs miRNAs is an endogenous non-coding single-stranded small RNAs composed of 21-23 bases, which can regulate the expression of genes at post-transcriptional level. MiRNAs are involved in many pathophysiological processes. At present, miRNAs is involved in myocardial ischemia. The study of function and mechanism in this paper is still in its infancy. In particular, there were few reports of miRNAs changes in the early stage of myocardial ischemia and no analysis of miRNA in cardiomyocytes after the early intervention of LVAD in the early stage of AMI. In order to provide theoretical basis for the treatment of acute myocardial infarction (AMI) with LVAD, this paper attempts to establish the expression profile of miRNA on the model of acute myocardial infarction. 1. Adult small tail Han sheep model of myocardial infarction was established by ligation of coronary artery. The heart was implanted with FW-2 type axial flow pump for control group and load relief group. After the model was established, electrocardiogram (ECG) was detected, and Swan-gaze floating catheter was inserted to record the results for 72 hours. The myocardial tissues of the control group and the load relief group were taken respectively after 72 hours. Divided into normal area, infarct side zone and infarct area, The apoptosis of cardiomyocytes was detected by tissue staining, Tunel method and high throughput sequencing technique. The expression profile of miRNA was established by high throughput sequencing. The results were verified by comparing the differentially expressed miRNA.7mRNAs in the corresponding regions of the control group and the unloading group. The functional and signal pathways of differentially expressed miRNA were analyzed. Among them, 3 rats were successfully made. LVAD group had 5 animals, among them, 3 rats were successfully stained with HE. The results of HE staining showed that the myocardial cell destruction in the unloading group was weaker than that in the control group. At the same time, the infiltration of inflammatory cells was significantly decreased. Masson staining showed that the area of collagen was decreased and the cardiac myocytes were retained more in the load relief group. The results of sirius red staining showed that the collagenization of the load relief group was significantly reduced. The results of Tunel showed that the apoptosis of myocardial cells was significantly decreased after load relief. The results showed that the apoptosis of cardiac myocytes in the unloaded group was significantly lower than that in the control group, and that in the unloaded group was significantly lower than that in the control group, and that in the unloaded group was significantly lower than that in the control group. The differential expression of miRNA in myocardial infarction region was oar-miR-19b (upregulation; infarct sideband region, miRNA was down-regulated by oar-miR41 1b-5p and oar-miR-487a-5p; Oar-miR-26a.5p was up-regulated; Oar-miR-19bP0.05) and oar-miR-26aP0.01 were significantly different between the unloading group and the control group. The results of .6go and KEGG analysis showed that the function and signal pathway involved in oar-miR-19b and oar-miR-26a were closely related to cardiovascular disease. Conclusion in this experiment, FW-2 type axial flow pump was successfully used to treat acute myocardial infarction in sheep. 3 days of circulatory assistance, It was proved that LVAD could effectively reduce the degree of myocardial infarction. The miRNA expression profiles of acute myocardial infarction and LVAD intervention were successfully established and verified. The results showed that the expression profile could be used to explore unknown miRNAs and to study the regulatory mechanism of their involvement in acute myocardial infarction response.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R542.22;R-332

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