CARD9在心肌缺血再灌注损伤中的作用及机制研究

发布时间：2018-04-16 21:42

本文选题：心肌缺血再灌注 + 炎症反应　；参考：《第四军医大学》2016年博士论文

【摘要】：研究背景冠心病是威胁人类健康的重大疾病,据《中国卫生和计划生育年鉴2013版》统计,我国2012年心血管疾病造成的死亡率为13.164/万,占全部疾病导致死亡总数的21.45%。其中因缺血性心血管疾病导致的死亡占心血管疾病总死亡率的93.17%。目前临床治疗缺血性心脏病的常规血管再灌注治疗方式包括药物溶栓治疗、经皮冠状动脉介入治疗术、冠状动脉旁路移植术等,挽救了大量缺血性心脏病患者的生命。但是,伴随治疗过程发生的缺血再灌注损伤(ischemia-reperfusion injury,IRI)越来越引起重视,无论围术期还是远期预后都可能对患者造成进一步的危害,导致远期心血管不良事件的再次发生。因此,临床迫切需要探索新的干预靶点和策略防治缺血再灌注损伤。心肌缺血再灌注损伤(Myocardial ischemia-reperfusion injury,MIRI)的发生机制涉及多种病理生理行为,其中炎症反应是MIRI中重要环节。缺血再灌注损伤会激活固有免疫应答,从而造成过度的炎症反应。过度的炎症反应导致活性氧簇(Reactive Oxygen Species,ROS)的分泌、免疫细胞的浸润、血管内皮的损伤,进而损伤心肌组织。而心肌梗死后期,炎症反应参与了组织修复。多项以炎症反应为治疗靶点的临床实验以失败告终,其可能的原因是非选择性的阻断或抑制炎症反应。胱天蛋白酶募集域蛋白9(Caspase Recruitment Domain containing protein 9,CARD9)是迄今为止发现的最重要的免疫衔接蛋白之一,存在于中性粒细胞、巨噬细胞等骨髓来源的免疫细胞中。既往报道,CARD9缺陷的小鼠或者人类,表现出对真菌等病原体的抵抗能力下降。研究表明,敲除CARD9会导致巨噬细胞分泌细胞因子能力的下降。在血管紧张素II诱导的高血压心脏病模型中,敲除CARD9可减少血管紧张素II对心功能的影响。我们前期研究证明,在高脂喂养的肥胖模型中,敲除CARD9可通过减弱炎症反应进而降低肥胖对心脏功能的影响。而CARD9在心肌缺血再灌注损伤中的作用尚不清楚。是否可以通过调控CARD9,进而影响MIRI中的炎症反应,最终达到降低心肌缺血再灌注损伤的效果尚不清楚。因此,我们在本课题中以此为科学假说开展研究,以期证明CARD9在心肌缺血再灌注损伤的作用及其可能的机制,并为MIRI的有效干预提供新的分子靶点。研究目的1.明确CARD9基因敲除是否会降低缺血再灌注损伤2.阐明CARD9基因通过何种方式影响缺血再灌注后心肌损伤3.明确CARD9参与缺血再灌注调控的上下游具体分子机制研究方法1.CARD9敲除对缺血再灌注后心肌损伤的作用研究通过基因筛选鉴定CARD9-/-小鼠,制备小鼠心肌缺血及心肌缺血再灌注模型,Western blot检测CARD9在心脏中的表达,小动物超声检测及血流动力学检测小鼠心脏功能,Evens blue/TTC双染法检测小鼠心肌缺血再灌注后心肌梗死面积,TUNEL染色测定小鼠心肌细胞凋亡率。2.CARD9基因参与氧化应激调节缺血再灌注后心肌损伤的在体研究制备小鼠心肌缺血及心肌缺血再灌注模型,通过Gr-1免疫荧光染色检测中性粒细胞的浸润,通过DCF荧光探针检测ROS的浸润,通过ELISA试剂盒检测心肌组织和血清中的TNF-α、IL-6、MCP-1以及CXCL-1的含量,通过Western blot检测心肌组织p38及p65的表达。3.CARD9通过NOD2-MAPK调控中性粒细胞炎症反应参与心肌缺血再灌注损伤的分子机制研究a)分离小鼠腹腔来源的中性粒细胞b)将分离纯化的中性粒细胞与H9c2心肌细胞共培养,加入MDP以激活CARD9c)通过TUNEL染色检测共培养心肌的凋亡情况d)通过ELISA试剂盒检测中性粒细胞上清中的TNF-α、IL-6、MCP-1以及CXCL-1的含量e)通过Western blot检测中性粒细胞中CARD9、p38及p65的表达研究结果1.敲除CARD9可减少小鼠心肌缺血再灌注后的心肌损伤a)缺血再灌注损伤后CARD9的表达上调b)敲除CARD9改善了小鼠心肌缺血再灌注损伤后的LVEF,LVEDP,+dp/dtmax和-dp/dtmax c)敲除CARD9减少了缺血再灌注损伤后的心肌梗死面积及细胞凋亡2.敲除CARD9减少了心肌缺血再灌所导致的炎症反应a)在缺血再灌注损伤后,与野生型小鼠相比,敲除CARD9减少了心肌缺血再灌所导致的中性粒细胞浸润b)在缺血再灌注损伤后,与野生型小鼠相比,敲除CARD9减少了心肌缺血再灌所导致的氧化应激,心肌组织中ROS的含量c)敲除CARD9减少了心肌组织及血清中由于缺血再灌所导致的TNF-α、IL-6、MCP-1以及CXCL-1细胞因子的分泌d)与野生型小鼠相比,CARD9敲除小鼠缺血再灌注损伤后心肌组织中p38、p65磷酸化水平降低3.CARD9通过NOD2-MAPK影响中性粒细胞的炎症反应a)与野生型中性粒细胞相比,敲除CARD9减少中性粒细胞造成的心肌细胞凋亡b)与野生型中性粒细胞相比,敲除CARD9减少降低细胞上清中,中性粒细胞分泌的TNF-α、IL-6、MCP-1以及CXCL-1的含量c)加入MDP可上调CARD9;与野生型中性粒细胞相比,敲除CARD9降低中性粒细胞的p38磷酸化,而不影响p65的磷酸化水平研究结论1.CARD9在MIRI后心肌组织中含量的升高,且这种升高与中性粒细胞浸润有关;敲除CARD9可以改善MIRI后心脏功能的损害、减少梗死面积以及凋亡细胞;2.CARD9通过NOD2-MAPK而不是NOD2-NFκB通路介导中性粒细胞参与的炎症反应,而其在MIRI环境中,可能还参与了其他PRRs介导的炎症反应。
[Abstract]:Background: coronary heart disease is a major threat to human health, health and family planning according to the < Chinese Yearbook > 2013 edition 2012 statistics, cardiovascular disease caused by China's mortality rate was 13.164/ million, accounted for 21.45%. of the total number of disease and death due to ischemic cardiovascular disease deaths accounted for total cardiovascular mortality at conventional clinical vascular 93.17%. the treatment of ischemic heart disease and reperfusion therapy including thrombolysis, percutaneous coronary intervention, coronary artery bypass grafting, save a large number of patients with ischemic heart disease in life. However, with treatment during ischemia reperfusion injury (ischemia-reperfusion, injury, IRI) have attracted more and more attention, whether perioperative or a long-term prognosis may cause further harm to the patients, leading to recurrence of long-term adverse cardiovascular events. Therefore, there is an urgent need to explore clinical intervention targets and strategy for prevention and treatment of ischemia reperfusion injury. The myocardial ischemia reperfusion injury (Myocardial ischemia-reperfusion, injury, MIRI) the occurrence mechanism involves the pathological and physiological behaviors of various, in which the inflammatory response is an important link in MIRI. Ischemia reperfusion injury activates the innate immune response, resulting in excessive inflammatory reaction.. excessive inflammation leads to reactive oxygen species (Reactive Oxygen, Species, ROS) the secretion of infiltrating immune cells, vascular endothelial injury, and myocardial injury and myocardial infarction. Later, inflammation is involved in tissue repair. A number of clinical trials for inflammation therapeutic targets failed, it may the reason is that the non selective blocking or inhibiting inflammation. Caspase recruitment domain protein 9 (Caspase Recruitment Domain containing protein 9, CARD9) Is by far the most important immune adaptor protein one exists in neutrophils, macrophages and other immune cells from bone marrow. Previous reports, CARD9 deficient mice or humans, showed a decrease in resistance to fungal pathogens. The results show that knockdown of CARD9 leads to the decrease of cytokine secretion ability of macrophages in angiotensin II induced hypertensive heart disease model, knockdown of CARD9 reduced angiotensin II effects on cardiac function. Our previous studies have demonstrated that in high fat fed obese model, knockdown of CARD9 can attenuate the inflammatory response and reduce the impact of obesity on cardiac function and the role of CARD9 in. Myocardial ischemia-reperfusion injury is not clear. Whether it can be regulated by CARD9, thereby affecting the inflammatory response in MIRI, will reduce the effect of myocardial ischemia reperfusion injury Is not clear. Therefore, we in this project as a scientific hypothesis research, in order to demonstrate the role of CARD9 in myocardial ischemia reperfusion injury and its possible mechanism, and provide new molecular targets for the effective intervention of MIRI. Objective: 1. specific CARD9 knockout 1.CARD9 research method will reduce reperfusion to clarify the effects of CARD9 gene on ischemic injury in 2. by the way in which 3. clear CARD9 myocardial reperfusion injury after ischemia reperfusion in ischemia on the downstream regulation of the molecular mechanism of knockout on ischemia reperfusion injury of myocardial damage induced by gene screening and identification of CARD9-/- mice, preparation of myocardial ischemia and myocardial ischemia reperfusion in mice model, the expression of Western blot detection of CARD9 in the heart, small animal ultrasound and hemodynamic detection of mouse cardiac function, Evens blue/TTC double staining assay in mice with myocardial ischemia reperfusion After the injection of myocardial infarction area was measured by TUNEL staining, myocardial injury of mice myocardial cell apoptosis rate of.2.CARD9 gene involved in oxidative stress regulation after ischemia reperfusion in vivo preparation of myocardial ischemia and myocardial ischemia-reperfusion model in mice, infiltration of neutrophils was detected by Gr-1 staining, the infiltration of DCF fluorescent probe for the detection of ROS by ELISA kit for detection of myocardial tissue and serum of TNF- alpha, IL-6, MCP-1 and CXCL-1 content, molecular mechanism of.3.CARD9 expression by Western blot and p65 p38 were detected by NOD2-MAPK regulation of neutrophilic inflammation in myocardial ischemia reperfusion injury a) neutrophil separation from mouse peritoneal B source) the separation and purification of neutrophils and H9c2 cells were co cultured, adding MDP to activate CARD9c) detection of co cultured myocardial apoptosis by D TUNEL staining ) by ELISA kit for detection of neutrophils in the supernatant of TNF- alpha, IL-6, MCP-1 and CXCL-1) by CARD9 Western blot E in neutrophils was detected, the expression of p38 and p65 1. knockdown of CARD9 can reduce myocardial ischemia reperfusion myocardial damage of mice after a) after ischemia reperfusion injury CARD9 the expression of B) knockout CARD9 improves myocardial ischemia reperfusion injury in mice after LVEF, LVEDP, +dp/dtmax and -dp/dtmax C) knockdown of CARD9 reduced ischemia reperfusion injury after myocardial infarction area and apoptosis cells 2. knockout inflammation CARD9 reduces myocardial ischemia reperfusion in ischemia caused by a) after reperfusion, compared with wild-type mice, knockout neutrophils CARD9 reduces myocardial ischemia reperfusion caused by infiltration of B) in ischemia reperfusion injury, compared with wild-type mice, knockdown of CARD9 reduced myocardial ischemia reperfusion by Oxidative stress leads to the content of myocardial ROS C) knockdown of CARD9 reduced the myocardial tissue and serum due to ischemia reperfusion caused by TNF- alpha, IL-6, MCP-1 and CXCL-1 cytokines d) compared with wild-type mice, CARD9 knockout mice after ischemia reperfusion injury of myocardial tissue in p38. The phosphorylation level of p65 decreased by 3.CARD9 NOD2-MAPK on inflammatory response of neutrophils a) compared with wild-type neutrophils, knockdown of myocardial cell apoptosis CARD9 neutropenia caused by B) compared with wild-type neutrophils, knockdown of CARD9 decreased cell supernatant, secretion of neutrophil TNF- alpha. IL-6, MCP-1 and CXCL-1 content C) MDP can increase CARD9; compared with wild-type neutrophils, knockdown of CARD9 decreased the phosphorylation of p38 in neutrophils, and does not affect the study of phosphorylation of p65 on 1.CARD9 after MIRI in the heart Increased content of muscle tissue, and this is associated with an increase in neutrophil infiltration; knockdown of CARD9 can improve the cardiac function after MIRI injury, reduce infarction area and apoptosis cells; inflammatory reaction by 2.CARD9 NOD2-MAPK instead of NOD2-NF B pathway mediated neutrophil involvement, and in the MIRI environment, may in other inflammatory reaction mediated by PRRs.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.4

【相似文献】