本文選題：心肌缺血再灌注 + 炎癥反應(yīng)　； 參考：《第四軍醫(yī)大學(xué)》2016年博士論文

【摘要】：研究背景冠心病是威脅人類健康的重大疾病,據(jù)《中國衛(wèi)生和計(jì)劃生育年鑒2013版》統(tǒng)計(jì),我國2012年心血管疾病造成的死亡率為13.164/萬,占全部疾病導(dǎo)致死亡總數(shù)的21.45%。其中因缺血性心血管疾病導(dǎo)致的死亡占心血管疾病總死亡率的93.17%。目前臨床治療缺血性心臟病的常規(guī)血管再灌注治療方式包括藥物溶栓治療、經(jīng)皮冠狀動(dòng)脈介入治療術(shù)、冠狀動(dòng)脈旁路移植術(shù)等,挽救了大量缺血性心臟病患者的生命。但是,伴隨治療過程發(fā)生的缺血再灌注損傷(ischemia-reperfusion injury,IRI)越來越引起重視,無論圍術(shù)期還是遠(yuǎn)期預(yù)后都可能對(duì)患者造成進(jìn)一步的危害,導(dǎo)致遠(yuǎn)期心血管不良事件的再次發(fā)生。因此,臨床迫切需要探索新的干預(yù)靶點(diǎn)和策略防治缺血再灌注損傷。心肌缺血再灌注損傷(Myocardial ischemia-reperfusion injury,MIRI)的發(fā)生機(jī)制涉及多種病理生理行為,其中炎癥反應(yīng)是MIRI中重要環(huán)節(jié)。缺血再灌注損傷會(huì)激活固有免疫應(yīng)答,從而造成過度的炎癥反應(yīng)。過度的炎癥反應(yīng)導(dǎo)致活性氧簇(Reactive Oxygen Species,ROS)的分泌、免疫細(xì)胞的浸潤、血管內(nèi)皮的損傷,進(jìn)而損傷心肌組織。而心肌梗死后期,炎癥反應(yīng)參與了組織修復(fù)。多項(xiàng)以炎癥反應(yīng)為治療靶點(diǎn)的臨床實(shí)驗(yàn)以失敗告終,其可能的原因是非選擇性的阻斷或抑制炎癥反應(yīng)。胱天蛋白酶募集域蛋白9(Caspase Recruitment Domain containing protein 9,CARD9)是迄今為止發(fā)現(xiàn)的最重要的免疫銜接蛋白之一,存在于中性粒細(xì)胞、巨噬細(xì)胞等骨髓來源的免疫細(xì)胞中。既往報(bào)道,CARD9缺陷的小鼠或者人類,表現(xiàn)出對(duì)真菌等病原體的抵抗能力下降。研究表明,敲除CARD9會(huì)導(dǎo)致巨噬細(xì)胞分泌細(xì)胞因子能力的下降。在血管緊張素II誘導(dǎo)的高血壓心臟病模型中,敲除CARD9可減少血管緊張素II對(duì)心功能的影響。我們前期研究證明,在高脂喂養(yǎng)的肥胖模型中,敲除CARD9可通過減弱炎癥反應(yīng)進(jìn)而降低肥胖對(duì)心臟功能的影響。而CARD9在心肌缺血再灌注損傷中的作用尚不清楚。是否可以通過調(diào)控CARD9,進(jìn)而影響MIRI中的炎癥反應(yīng),最終達(dá)到降低心肌缺血再灌注損傷的效果尚不清楚。因此,我們?cè)诒菊n題中以此為科學(xué)假說開展研究,以期證明CARD9在心肌缺血再灌注損傷的作用及其可能的機(jī)制,并為MIRI的有效干預(yù)提供新的分子靶點(diǎn)。研究目的1.明確CARD9基因敲除是否會(huì)降低缺血再灌注損傷2.闡明CARD9基因通過何種方式影響缺血再灌注后心肌損傷3.明確CARD9參與缺血再灌注調(diào)控的上下游具體分子機(jī)制研究方法1.CARD9敲除對(duì)缺血再灌注后心肌損傷的作用研究通過基因篩選鑒定CARD9-/-小鼠,制備小鼠心肌缺血及心肌缺血再灌注模型,Western blot檢測CARD9在心臟中的表達(dá),小動(dòng)物超聲檢測及血流動(dòng)力學(xué)檢測小鼠心臟功能,Evens blue/TTC雙染法檢測小鼠心肌缺血再灌注后心肌梗死面積,TUNEL染色測定小鼠心肌細(xì)胞凋亡率。2.CARD9基因參與氧化應(yīng)激調(diào)節(jié)缺血再灌注后心肌損傷的在體研究制備小鼠心肌缺血及心肌缺血再灌注模型,通過Gr-1免疫熒光染色檢測中性粒細(xì)胞的浸潤,通過DCF熒光探針檢測ROS的浸潤,通過ELISA試劑盒檢測心肌組織和血清中的TNF-α、IL-6、MCP-1以及CXCL-1的含量,通過Western blot檢測心肌組織p38及p65的表達(dá)。3.CARD9通過NOD2-MAPK調(diào)控中性粒細(xì)胞炎癥反應(yīng)參與心肌缺血再灌注損傷的分子機(jī)制研究a)分離小鼠腹腔來源的中性粒細(xì)胞b)將分離純化的中性粒細(xì)胞與H9c2心肌細(xì)胞共培養(yǎng),加入MDP以激活CARD9c)通過TUNEL染色檢測共培養(yǎng)心肌的凋亡情況d)通過ELISA試劑盒檢測中性粒細(xì)胞上清中的TNF-α、IL-6、MCP-1以及CXCL-1的含量e)通過Western blot檢測中性粒細(xì)胞中CARD9、p38及p65的表達(dá)研究結(jié)果1.敲除CARD9可減少小鼠心肌缺血再灌注后的心肌損傷a)缺血再灌注損傷后CARD9的表達(dá)上調(diào)b)敲除CARD9改善了小鼠心肌缺血再灌注損傷后的LVEF,LVEDP,+dp/dtmax和-dp/dtmax c)敲除CARD9減少了缺血再灌注損傷后的心肌梗死面積及細(xì)胞凋亡2.敲除CARD9減少了心肌缺血再灌所導(dǎo)致的炎癥反應(yīng)a)在缺血再灌注損傷后,與野生型小鼠相比,敲除CARD9減少了心肌缺血再灌所導(dǎo)致的中性粒細(xì)胞浸潤b)在缺血再灌注損傷后,與野生型小鼠相比,敲除CARD9減少了心肌缺血再灌所導(dǎo)致的氧化應(yīng)激,心肌組織中ROS的含量c)敲除CARD9減少了心肌組織及血清中由于缺血再灌所導(dǎo)致的TNF-α、IL-6、MCP-1以及CXCL-1細(xì)胞因子的分泌d)與野生型小鼠相比,CARD9敲除小鼠缺血再灌注損傷后心肌組織中p38、p65磷酸化水平降低3.CARD9通過NOD2-MAPK影響中性粒細(xì)胞的炎癥反應(yīng)a)與野生型中性粒細(xì)胞相比,敲除CARD9減少中性粒細(xì)胞造成的心肌細(xì)胞凋亡b)與野生型中性粒細(xì)胞相比,敲除CARD9減少降低細(xì)胞上清中,中性粒細(xì)胞分泌的TNF-α、IL-6、MCP-1以及CXCL-1的含量c)加入MDP可上調(diào)CARD9;與野生型中性粒細(xì)胞相比,敲除CARD9降低中性粒細(xì)胞的p38磷酸化,而不影響p65的磷酸化水平研究結(jié)論1.CARD9在MIRI后心肌組織中含量的升高,且這種升高與中性粒細(xì)胞浸潤有關(guān);敲除CARD9可以改善MIRI后心臟功能的損害、減少梗死面積以及凋亡細(xì)胞;2.CARD9通過NOD2-MAPK而不是NOD2-NFκB通路介導(dǎo)中性粒細(xì)胞參與的炎癥反應(yīng),而其在MIRI環(huán)境中,可能還參與了其他PRRs介導(dǎo)的炎癥反應(yīng)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R541.4

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