乙醛脱氢酶2在动脉粥样硬化泡沫细胞中的作用研究

发布时间：2018-06-29 01:03

本文选题：乙醛脱氢酶2 + 动脉粥样硬化　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景动脉粥样硬化是心、脑血管和外周血管疾病中首要的发病病因。动脉粥样硬化由高血压、高血脂、吸烟、糖尿病等多种危险因素作用引起,其病理生理的发生发展过程复杂,其中脂质代谢障碍与紊乱公认为动脉粥样硬化病理学基础。在动脉粥样硬化初始发病过程中,脂质沉积于血管壁,巨噬细胞募集并进入血管内皮下基质吞噬脂质,从而分化为泡沫细胞,形成早期动脉粥样硬化斑块中的脂质条纹。在动脉粥样硬化晚期,泡沫细胞发生坏死或凋亡,造成脂质外泄而形成坏死核心,增加斑块的不稳定性。所以,泡沫细胞的发生与发展贯穿动脉粥样硬化的病理生理过程。乙醛脱氢酶2(Aldehyde dehydrogenase 2,ALDH2)是定位在线粒体上的一种醛类物质代谢酶,野生型的ALDH2酶活性高而突变型的ALDH2酶活性降低或失活。近年来,流行病学研究显示ALDH2参与至急性动脉综合征和冠状动脉粥样硬化性心脏病中。来自日本、中国和韩国的几项研究证明ALDH2突变型与心肌梗死、急性冠脉综合症等心血管疾病紧密相关。在2012年,一项全基因组关联分析对ALDH2基因多态性和冠心病进行研究,证实突变型ALDH2的人群高发冠脉综合症和冠状动脉粥样硬化性疾病。既往研究表明ALDH2在内皮细胞和心肌细胞保护等方面发挥重要的作用,但是ALDH2在泡沫细胞中的作用尚未有相关研究报道。在本课题组的前期研究中,我们发现利用慢病毒敲除ALDH2的小鼠表现出更多的颈动脉动脉粥样硬化斑块,其中脂质沉积明显增多。在本实验中,我们同样证实了 ALDH2敲除小鼠在主动脉根部也表现出更多的脂质沉积。由于泡沫细胞是动脉粥样硬化斑块中脂质沉积必不可缺的一部分,同时,近几年来ALDH2的研究都表明其在心血管系统上发挥着重要的作用,所以我们假设ALDH2在泡沫细胞中发挥了重要的作用并依此在动物体内和体外的相关验证实验。研究目的1.研究ALDH2对动脉粥样硬化脂质沉积的影响。2.探讨ALDH2在泡沫细胞中调控脂质沉积的机制,为动脉粥样硬化的病理生理过程提供进一步的理论支持。研究方法1.乙醛脱氢酶2缺乏小鼠的脂质沉积的变化1.1 ALDH2基因缺乏增加动脉粥样硬化斑块中的脂质ApoE-/-小鼠高脂喂养构建动脉粥样硬化模型,利用慢病毒干扰小鼠ALDH2表达。把老鼠随机分组分为ALDH2缺乏的实验组与ALDH2正常的对照组。取小鼠主动脉根部动脉粥样硬化斑块制成冰冻切片,并用油红O染色检测脂质含量。1.2ALDH2影响泡沫细胞的形成1.2.1 ALDH2敲基因小鼠腹腔巨噬细胞的提取与鉴定利用ALDH2敲基因小鼠与野生小鼠作为巨噬细胞供体,在thioglycollate刺激下提取小鼠腹腔原代巨噬细胞。用细胞免疫荧光方法鉴定腹腔巨噬细胞与ALDH2的表达情况。1.2.2 ALDH2缺乏对泡沫细胞形成的影响小鼠原代巨噬细胞分为ALDH2缺乏组(ALDH2-/-)与对照组(ALDH2+/+)。分别加入氧化低密度脂蛋白刺激后,利用油红O进行脂质染色,镜下观察并计数泡沫细胞形成。1.2.3 ALDH2缺乏对泡沫细胞内脂质沉积的影响小鼠原代巨噬细胞加入氧化低密度脂蛋白作用后,利用胆固醇试剂盒,通过测量胆固醇吸光度检测泡沫细胞内胆固醇含量。2.ALDH2对泡沫细胞脂质代谢的作用2.1 ALDH2对脂质摄入相关受体的蛋白表达的影响Western Blot方法测量ox-LDL处理后ALDH2-、-与ALDH2+/+泡沫细胞主要的脂质摄入受体CD36、LOX-1、SRA的蛋白表达变化。2.2 ALDH2对脂质流出相关受体的蛋白表达的影响Western Blot方法测量Ox-LDL处理后ALDH2-/-与ALDH2+/+泡沫细胞主要的脂质摄入受体ABCA1、ABCG1、ACAT-1的蛋白表达变化。3.ALDH2对CD36转录调控的作用的影响3.1 CD36在泡沫细胞形成中的作用ALDH2-/-与ALDH2+/+在ox-LDL处理前分别加入CD36受体抑制剂(Sulfo-N-succinimidyloleate,SSO),油红O镜下观察泡沫细胞的形成并计数。3.2抑制CD36对泡沫细胞中脂质的影响ALDH2-/-与ALDH2+/+在ox-LDL处理前分别加入CD36受体抑制剂(Sulfo-N-succinimidyloleate,SSO),利用胆固醇试剂盒,通过测量胆固醇吸光度检测泡沫细胞内胆固醇含量。3.3 ALDH2缺乏下CD36的mRNA变化Trizol 方法提取 ox-LDL 处理后 ALDH2-/-与ALDH2+/+的总 RNA,RT-PCR 方法检测CD36 mRNA的水平。3.4 ALDH2影响CD36转录调控因子PPARyWestern Blot方法测量ox-LDL处理后ALDH2-/-与ALDH2+/+泡沫细胞中CD36的转录调控因子PPARy的蛋白水平。4.ALDH2对泡沫细胞凋亡的作用4.1 ALDH2对泡沫细胞凋亡的影响Tunel法观察Ox-LDL处理后ALDH2/-与ALDH2+/+泡沫细胞凋亡情况,Western Blot方法检测促凋亡蛋白Caspase-3的蛋白表达水平。4.2 ALDH2对毒性醛类物质的影响利用丙二醛检测试剂盒,通过测量丙二醛吸光度检测泡沫细胞内丙二醛含量。Western Blot方法检测4-羟基壬烯醛对泡沫细胞内全蛋白的加聚影响。4.3 ALDH2对泡沫细胞凋亡相关通路的影响Western Blot方法检测调控凋亡的相关信号通路蛋白p-AKT/AKT、p-P38/P38 的变化。研究结果:1.ALDH2对乙醛脱氢酶2缺乏小鼠脂质沉积的影响1.1 ALDH2基因缺乏增加动脉粥样硬化斑块中的脂质慢病毒干扰下乙醛脱氢酶2缺乏的APOE-/-组与正常APOE-/-组的主动脉根部动脉粥样硬化斑块脂质沉积增加。1.2ALDH2影响泡沫细胞的形成1.2.1 ALDH2敲基因小鼠腹腔巨噬细胞的提取与鉴定免疫荧光显示ALDH2-/-小鼠提取的原代腹腔细胞为ALDH2缺乏的巨噬细胞,ALDH2+/+小鼠提取的原代腹腔细胞正常表达ALDH2的巨噬细胞。1.2.2 ALDH2缺乏下抑制泡沫细胞形成ALDH2-/-组巨噬细胞与ALDH2+/+在氧化低密度脂蛋白刺激后都形成了泡沫细胞。其中ALDH2-/-泡沫细胞形成多于ALDH2+/+组。1.2.3 ALDH2缺乏的泡沫细胞内脂质沉积减少ALDH2-/-泡沫细胞中的胆固醇含量多于ALDH2+/+组。2.ALDH2对泡沫细胞脂质代谢的影响2.1 ALDH2对脂质摄入相关受体的蛋白表达Ox-LDL处理后,ALDH2-/-泡沫细胞与ALDH2+/+泡沫细胞相比,CD36蛋白表达量下降。LOX-1、SRA的蛋白表达无明显变化。2.2 ALDH2对脂质流出相关受体的蛋白表达Ox-LDL处理后,ALDH2-/-泡沫细胞与ALDH2+/+泡沫细胞相比,ABCA 1、ABCG1、ACAT-1蛋白表达无明显变化。3.ALDH2对CD36转录调控的作用3.1 CD36在泡沫细胞形成中的作用加入CD36受体抑制剂后ALDH2-/-与ALDH2+/+泡沫细胞形成无明显差异。3.2抑制CD36对泡沫细胞中脂质的影响加入CD36受体抑制剂后ALDH2-/-与ALDH2+/+泡沫细胞内胆固醇含量无明显差异。3.3 ALDH2缺乏下CD36的mRNA变化Ox-LDL处理后,ALDH2-/-泡沫细胞与ALDH2+/+泡沫细胞相比,CD36 mRNA的表达水平下降。3.4 ALDH2影响CD36转录调控因子PPAR γOx-LDL处理后,ALDH2-/-泡沫细胞与ALDH2+/+泡沫细胞相比,CD36的转录调控因子PPARy的蛋白水平下降。4.ALDH2对泡沫细胞凋亡的作用4.1 ALDH2缺乏促进泡沫细胞凋亡高浓度ox-LDL处理后ALDH2-/-与ALDH2+/+相比泡沫细胞发生更多的凋亡,促凋亡蛋白Caspase-3的蛋白表达水平上调。4.2 ALDH2缺乏下毒性醛类物质增加高浓度ox-LDL处理后ALDH2-/-与ALDH2+/+相比泡沫细胞内丙二醛含量增加,4-羟基壬烯醛对泡沫细胞内全蛋白的加聚增多。4.3 ALDH2对泡沫细胞调亡相关通路的影响高浓度ox-LDL处理后ALDH2-/-与ALDH2+/+相比下,调控凋亡的信号通路蛋白p-AKT/AKT激活、调控增殖的信号通路蛋白p-P38/P38抑制。研究结论与意义1.首次发现ALDH2的缺乏促进泡沫细胞内毒性醛的增加,通过抑制脂质摄取受体蛋白CD36的转录调控因子PPARγ下调CD36的蛋白表达,从而减少泡沫细胞的脂质摄取。2.首次揭示ALDH2缺乏的泡沫细胞激活凋亡信号通路p-AKT/AKT、抑制增殖信号通路p-P38/P38而引起泡沫细胞凋亡增加。
[Abstract]:Atherosclerosis is the primary cause of the disease in the heart, cerebrovascular and peripheral vascular diseases. Atherosclerosis is caused by many dangerous factors, such as hypertension, hyperlipidemia, smoking, diabetes and other dangerous factors, and its pathophysiological process is complicated. Lipid metabolism disorders and disorders are recognized as the pathological basis of atherosclerosis. In the process of initial atherosclerosis, lipids are deposited on the wall of the blood vessel, and macrophages raise and enter the endothelium to devour the lipid, thus differentiating into foam cells and forming lipid stripes in early atherosclerotic plaques. In the late stage of atherosclerosis, the necrosis or apoptosis of foam cells causes lipid release. It forms the core of necrosis and increases the instability of plaque. Therefore, the occurrence and development of the foam cells run through the pathophysiological process of atherosclerosis. Acetaldehyde dehydrogenase 2 (Aldehyde dehydrogenase 2, ALDH2) is an aldehyde metabolizing enzyme located on the mitochondria. The activity of the wild type ALDH2 enzyme is high and the mutant ALDH2 enzyme activity is reduced. In recent years, epidemiological studies have shown that ALDH2 is involved in acute arterial syndrome and coronary atherosclerotic heart disease. Several studies from Japan, China and South Korea have proved that ALDH2 mutants are closely related to cardiovascular diseases such as myocardial infarction and acute coronary syndrome. In 2012, a whole genome association analysis of AL DH2 gene polymorphism and coronary heart disease have been studied, which confirmed that the population of mutant ALDH2 has high incidence of coronary syndrome and coronary atherosclerotic disease. Previous studies have shown that ALDH2 plays an important role in the protection of endothelial cells and myocardial cells, but the role of ALDH2 in foam cells has not been reported. In the early study of the group, we found that mice using lentivirus knockout ALDH2 showed more carotid atherosclerotic plaques in which the lipid deposition was significantly increased. In this experiment, we also confirmed that ALDH2 knockout mice also showed more lipid deposition at the root of the aorta. Because the foam cells were atherosclerosis. There is an essential part of lipid deposition in plaque. At the same time, ALDH2 research has shown that it plays an important role in the cardiovascular system in recent years. So we assume that ALDH2 plays an important role in foamy cells and is related to the laboratory test in vivo and in vitro. Objective 1. to study ALDH2 for arterial porridge. The effect of sclerosing lipid deposition on the mechanism of ALDH2 in the regulation of lipid deposition in foam cells to provide further theoretical support for the pathophysiological process of atherosclerosis. Methods 1. acetaldehyde dehydrogenase 2 deficiency mice lipid deposition changes 1.1 ALDH2 genes lack of increased lipid ApoE-/- in atherosclerotic plaques Rats were fed by high fat feeding to construct the atherosclerotic model, using lentivirus to interfere with the expression of ALDH2 in mice. The rats were randomly divided into ALDH2 deficiency experimental group and normal ALDH2 control group. The atherosclerotic plaque of the aortic root of mice was made into frozen section, and the lipid content of.1.2ALDH2 was used to detect the effect of.1.2ALDH2 on the shape of the foam cells. Extraction and identification of peritoneal macrophages of 1.2.1 ALDH2 knockout mouse peritoneal macrophages, ALDH2 knockout mice and wild mice were used as macrophage donors and mouse peritoneal macrophages were extracted under thioglycollate stimulation. The expression of peritoneal macrophages and ALDH2 was identified by cell immunofluorescence method.1.2.2 ALDH2 deficiency on foams The primary macrophages of mice were divided into ALDH2 deficiency group (ALDH2-/-) and control group (ALDH2+/+). After addition of oxidized low density lipoprotein (LDL), lipid staining was carried out with oil red O. The effect of.1.2.3 ALDH2 deficiency on the lipid deposition of foam cell in the foam cells was observed under the microscope, and the primary macrophage of the mouse was added to the foam cell. After oxidation of low density lipoprotein (LDL), use Cholesterol Reagent box to measure the effect of cholesterol content.2.ALDH2 on the lipid metabolism of foam cells by measuring cholesterol absorbency. Effect of 2.1 ALDH2 on the protein expression of lipid intake related receptors by Western Blot method, ALDH2-, and ALDH2+/+ foam cells after ox-LDL treatment Major lipid intake receptor CD36, LOX-1, SRA protein expression changes,.2.2 ALDH2 effect on the protein expression of lipid efflux receptor, Western Blot method measured the main lipid intake receptor ABCA1 of ALDH2-/- and ALDH2+/+ foam cells after Ox-LDL treatment and the effect of protein expression changes on the regulation of transcription. The effect of 3.1 CD36 on the formation of foam cells ALDH2-/- and ALDH2+/+ were added to the CD36 receptor inhibitor (Sulfo-N-succinimidyloleate, SSO) before ox-LDL treatment. The formation of foam cells was observed under the oil red O microscope and the effect of.3.2 inhibition CD36 on the lipid in the foam cells was counted. Receptor inhibitor (Sulfo-N-succinimidyloleate, SSO), using Cholesterol Reagent box, the total RNA of ALDH2-/- and ALDH2+/+ after ox-LDL treatment was extracted by measuring the cholesterol content of.3.3 ALDH2 in foam cells by the measurement of cholesterol absorbency and the mRNA change Trizol method under the absence of CD36. The effect of transcription regulator PPARyWestern Blot on ALDH2-/- and ALDH2+/+ in ALDH2-/- and ALDH2+/+ foam cells the effect of protein level.4.ALDH2 on the apoptosis of foam cells 4.1 ALDH2 on the apoptosis of foam cells, the Tunel method was used to observe the apoptosis of ALDH2/- and foam cells after Ox-LDL treatment. Lot method was used to detect the protein expression level of apoptotic protein Caspase-3.4.2 ALDH2 on the toxicity of toxic aldehydes using malondialdehyde detection kit. By measuring malondialdehyde absorbance detection of malondialdehyde content in foam cells,.Western Blot method was used to detect the effect of 4- hydroxyl nonylaldehyde on the accumulation of whole protein in the foam cell by the addition of 4- hydroxyl nonylaldehyde to.4.3 ALDH2 pair of bubbles. Influence of Western Blot method on apoptosis related signaling pathway protein p-AKT/AKT and p-P38/P38 changes. Results: the effect of 1.ALDH2 on lipid deposition in acetaldehyde dehydrogenase 2 deficiency in mice; 1.1 ALDH2 gene deficiency increased glyaldehyde dehydrogenase 2 in atherosclerotic plaques Lipid deposition in atherosclerotic atherosclerotic plaques in the aortic root of the APOE-/- group and the normal APOE-/- group increased the effect of.1.2ALDH2 on the formation of the foam cells. The extraction and identification of the peritoneal macrophages in the 1.2.1 ALDH2 knockout mice, the immunofluorescence showed that the primary peritoneal cells extracted by ALDH2-/- mice were ALDH2 deficient macrophages, ALDH2+/+ mice The extracted primary peritoneal cells normally express ALDH2 macrophage.1.2.2 ALDH2 deficiency to inhibit the formation of foam cells and form ALDH2-/- group macrophages and ALDH2+/+ to form foam cells after oxidative low density lipoprotein stimulation. Among them, ALDH2-/- foam cells form more than the ALDH2+/+ group.1.2.3 ALDH2 deficient foam cells in the lipid deposition. The cholesterol content in the small ALDH2-/- foam cells was more than that of the ALDH2+/+ group.2.ALDH2 on the lipid metabolism of the foam cells. 2.1 ALDH2 was treated with the protein expression of the receptor related to the lipid intake. The ALDH2-/- foam cells were compared with the ALDH2+/+ foam cells, the expression of CD36 protein decreased.LOX-1, and the protein expression of SRA had no obvious change of.2.2 ALDH2. After Ox-LDL treatment of the protein expression of the lipid outflow related receptor, ALDH2-/- foam cells compared with ALDH2+/+ foam cells, ABCA 1, ABCG1, ACAT-1 protein expression did not significantly change the role of.3.ALDH2 on the regulation of CD36 transcriptional regulation. 3.1 CD36 in the formation of foam cells added to the CD36 receptor inhibitor, ALDH2-/- and ALDH2+/+ foam cells were formed. Significant difference in the effect of.3.2 on the effect of CD36 on the lipid in the foam cells. There is no significant difference in the cholesterol content between the ALDH2-/- and the ALDH2+/+ foam cells after the CD36 receptor inhibitor..3.3 ALDH2 lack of CD36's mRNA change Ox-LDL treatment, ALDH2-/- foam cells compared with the ALDH2+/+ foam cells CD36 transcriptional regulator PPAR gamma Ox-LDL treatment, ALDH2-/- foam cells compared with ALDH2+/+ foam cells, CD36 transcriptional regulator PPARy protein level decreased.4.ALDH2 to the apoptosis of foam cells 4.1 ALDH2 deficiency promoted the foam cell apoptosis high concentration ox-LDL processing, ALDH2-/- compared with ALDH2+/+ cells compared to ALDH2+/+ cells. Apoptosis, protein expression level of apoptotic protein Caspase-3 up regulate the increase of the content of malondialdehyde in foam cells after ALDH2-/- and ALDH2+/+, and the effect of 4- hydroxyl nonylaldehyde on the apoptosis of foam cells after.4.2 ALDH2 deficiency increases the concentration of ALDH2-/- and ALDH2+/+. The effect of 4- hydroxyl nonylaldehyde on the apoptosis of foam cells in foam cells After high concentration of ox-LDL, ALDH2-/- and ALDH2+/+ are compared with ALDH2+/+, the signaling pathway protein p-AKT/AKT regulates apoptosis and regulates the proliferation of signaling pathway protein p-P38/P38. Conclusion and significance 1. first found that the deficiency of ALDH2 promotes the increase of toxic aldehyde in foam cells and inhibits the transcriptional regulation of the lipid uptake receptor protein CD36. SubPPAR gamma down regulated the protein expression of CD36, thus reducing the lipid uptake by.2. in foam cells for the first time to reveal the p-AKT/AKT of the apoptotic signaling pathway in the ALDH2 deficient foam cells, and to inhibit the proliferation signal pathway p-P38/P38 and increase the apoptosis of the foam cells.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

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