脂联素对巨噬细胞胆固醇逆转运的影响及其机制

发布时间：2018-05-04 08:25

本文选题：脂联素 + 脂联素基因敲击小鼠　；参考：《山西医科大学》2017年博士论文

【摘要】：当前,在全世界范围内,心血管疾病的发病率和死亡率均位于前几位,对人们的身心健康有着重大威胁。目前我国罹患心血管病者约2.3亿人,其中约300万人因该病死亡。在心脑血管病变中动脉粥样硬化(Atherosclerosis,AS)为关键的病理基础,以胆固醇代谢异常为主的脂质代谢紊乱是重要的致病因素;该疾病最主要的病理特点是巨噬细胞能清除高水平的脂质产生泡沫细胞。基于以上背景,研究者们热衷于探讨动脉粥样硬化病变的发生发展机制、怎样避免形成泡沫细胞。胆固醇逆转运(Reverse Cholesterol ransport,RCT)指的是把细胞中含有的游离性胆固醇向胞外转移,并聚集至肝脏,最终排出机体;RCT全过程包括下列各步骤:最初外周细胞内含有大量胆固醇经转运蛋白(如ABCA1和ABCG1等)的介导进而结合于载脂蛋白,生成成熟状态的HDL,再随着血液系统聚集到肝脏,最后排出机体。ATP结合盒转运子(Binding Cassette Transporter,ABC)属于当前存在的数量最多的膜转运家族。其中ATP三磷酸腺苷结合盒转运子A1(ABCA1)是转运时功能最突出的转运蛋白,有助于细胞排出胆固醇。在ABCA1转运蛋白的作用下脂质可发生外流,再结合于apoA-I,进而产生一定水平的磷脂-apoA-I复合物,并且能够酯化细胞外游离状态的胆固醇,最终可生成成熟状态的HDL,随着血液系统聚集在肝脏中,进而能够被肝脏所利用而生成胆汁酸盐,和粪便排出体外[1-3]。所以,提升RCT水平有助于防止AS病变。相关的研究结果表明,高密度脂蛋白(High Density Lipoprotein Cholesterol,HDL-C)数量与机体发生动脉粥样硬化的可能性呈显著的负相关关系。HDL-C拮抗动脉粥样硬化的主要机制在于介导RCT过程。其中RCT能将外周组织内含有的大量胆固醇通过HDL-C转运到肝脏以胆汁酸的形式排出体外,是目前相关领域研究者们公认的抗动脉粥样硬化的关键机制。在RCT中ABCA1有利于细胞中磷脂、胆固醇向HDL-C前体apoA-I转运,形成新生HDL-C,进而启动RCT,所以ABCA1在RCT和HDL-C新生成中起着极其重要的起始限速作用。所以能否通过调节ABCA1蛋白的表达而达到促进胆固醇逆转运成为广受关注的抗动脉粥样硬化的靶点。脂联素(Adiponectin)主要来源于脂肪细胞,能够发挥胰岛素抵抗、抗动脉粥样硬化及抗炎等作用。研究表明:在肥胖、高血压、糖尿病、心肌受损、动脉粥样硬化等病人血清内具有较低水平的脂联素。并且,很多临床试验结果显示脂联素浓度明显降低与高血压、血管内皮受损、冠心病及心肌梗死等病变的发生发展存在紧密的联系[4]。在发生AS疾病的过程中,脂联素能够对血管平滑肌细胞的分化、繁殖进行抑制,并避免巨噬细胞不断转化为泡沫细胞,使脂联素在内皮细胞中发挥抗AS功效。脂联素对巨噬细胞胆固醇逆转运的影响及其机制在\在冠状动脉粥样硬化AS早期内皮细胞可被炎症细胞激活在血管壁上沉积并呈剂量依赖性抑制TNF诱导的在人体主动脉内皮上的细胞粘附作用当发生动脉硬化或其他大血管病变时大量的脂联素会聚集在受损的血管壁上而导致患者脂联素的消耗血浆脂联素随着动脉粥样硬化的发展呈进行性下降低脂联素血症是冠状动脉粥样硬化发展的独立危险因子脂联素缺乏小鼠在动脉发生损伤的时候表现为严重的内膜增厚使用外源性脂联素可使内膜增殖得到改善男性的脂联素往往低于女性有人认为这可能是女性冠心病发病率低于男性的一个原因最近发生过心血管疾病患者脂联素水平降低脂联素每升高1μg/ml心血管的危险性则下降3%脂联素可降低肿瘤坏死因子TNF-α刺激的粘附分子的表达抑制外周单核细胞的生长和炎症因子的释放具有保护内皮抗炎拮抗动脉粥样硬化的作用它的水平与甘油三酯载脂蛋白呈负相关与高密度脂蛋白及载脂蛋白A-I呈正相关。本课题通过细胞水平和动物水平两个部分来探讨脂联素对胆固醇逆转运的影响。选择脂联素基因敲除小鼠(Adiponectin-/-GeneKnockOutMice)作为研究动物,连续8周坚持高脂饲养,建立动脉粥样硬化模型。对脂联素组动物血脂水平改变情况进行检测;小鼠注射3H-TC后,经液闪计数仪对脂联素处理后动物肝脏、血清、粪便内含有的胆固醇流出量进行检测;经Western-blot、PCR法对脂联素处理后动物ABCA1的mRNA水平、蛋白量进行检测,深入的分析脂联素对RCT产生的影响和相关的作用机理,为临床上有效的防止疾病的发生发展给予一个新方向、新思路。1、脂联素通过肝X受体α信号通路上调RAW264.7巨噬细胞中ABCA1、ABCG1的表达及意义目的:离体研究脂联素APN是否是通过肝X受体α信号通路影响RAW264.7源性泡沫细胞内含有的ABCA1、ABCG1表达的mRNA水平、蛋白量、胆固醇流出量、胆固醇总水平。方法:培养RAW264.7巨噬细胞给予ox-LDL诱导为泡沫细胞。使用实时荧光定量PCR和westernblot检测ABCA1、ABCG1mRNA和蛋白的表达;液体闪烁计数测定荷脂THP-1巨噬细胞和RAW264.7巨噬细胞内胆固醇流出率;高效液相色谱法检测细胞内胆固醇含量。结果:脂联素通过LXRα信号通路上调RAW264.7 巨噬细胞中ABCA1、ABCG1表达。结论:脂联素通过LXRα途径提高RAW264.7巨噬细胞中的ABCA1、ABCG1表达和胆固醇流出。为预防和治疗动脉粥样硬化提供了理论基础。2、脂联素对APN-/-动脉粥样硬化小鼠ABCA1表达及动脉粥样硬化的影响目的:研究脂联素对脂联素基因敲除小鼠(APN-/-小鼠)体内巨噬细胞胆固醇逆转运的影响及其在抗动脉粥样硬化中可能的作用机制。1.观察脂联素对ApoE-/-小鼠颈动脉粥样硬化斑块的作用;2.探讨脂联素对斑块内硝基化应激的作用及其可能机制;3.观察脂联素对斑块局部血流剪切力的影响,探讨其抑制斑块的其他可能机制。方法:1.模型建立:选择4周龄脂联素基因敲除小鼠(Adiponectin-/-geneknockoutmice)30只,高脂喂养8周,建立动脉粥样硬化模型。2.分组:动物随机分为五组:空白对照组,50μg/(kg·d)干预组,150μg/(kg·d)干预组,200μg/(kg·d)干预组,250μg/(kg·d)干预组。3.方法:脂联素干预4周后,向APN-/-小鼠腹腔注射乙酰化LDL与3 H-TC标记的RAW264.7 巨噬细胞悬液,酶法测定小鼠血清血脂水平,液闪计数仪测定小鼠肝脏、血清及粪便中3H-TC占腹腔注射3H-TC总量的百分比,RT-PCR及Western-blot检测肝脏中ABCA1的mRNA及蛋白表达水平。结果:1.与对照组相比,脂联素干预组血清TG、TC、LDL水平明显降低,HDL升高;且此改变随脂联素呈剂量依赖性改变。2.对照组主动脉粥样硬化病变明显,光镜下可见血管壁有大量脂质沉积,有粥样斑块形成,有散在泡沫细胞;脂联素干预组APN-/-小鼠的主动脉粥样硬化病变程减轻,且随脂联素呈剂量依赖性改变。3.脂联素干预组血清3 H-TC含量明显降低(P0.05),肝脏及粪便中3 H-TC含量明显升高(P0.05)。4.RT-PCR检测结果显示:与对照组相比,脂联素干预组肝脏组织ABCA1的mRNA转录水平明显提高,且随脂联素呈剂量依赖性增加(P0.05)。5.Western-blot结果显示:与对照组相比,使用脂联素干预的实验组肝脏组织ABCA1的蛋白表达水平明显提高,且随脂联素呈剂量依赖性增加(P0.05)。结论:1.脂联素能够降低血清TC、TG及LDL水平,提高HDL水平;2.脂联素可以减轻小鼠主动脉粥样硬化;3.脂联素可以增加血清胆固醇流出率;4.脂联素能够呈剂量依赖性上调肝脏组织中ABCA1的mRNA及蛋白表达。脂联素通过上调ABCA1来促进胆固醇逆转运,促进细胞内胆固醇流出,从而延缓动脉粥样硬化的发生及发展,为心血管疾病的防治提供了新思路。
[Abstract]:At present, in the world, the incidence and mortality of cardiovascular diseases are located in the first few places, and there is a major threat to people's physical and mental health. At present, about 230 million people with cardiovascular disease in China, about 3 million of them died of the disease. The Atherosclerosis (AS) is the key pathological basis in cardiovascular and cerebrovascular diseases. The lipid metabolism disorder characterized by abnormal cholesterol metabolism is an important pathogenic factor; the most important pathological feature of the disease is that macrophages can remove high levels of lipid producing foam cells. Based on the above background, researchers are keen to explore the mechanism of the development of atherosclerotic lesions and how to avoid foamy cells. Reverse Cholesterol ransport (RCT) refers to the transfer of free cholesterol in cells to the cell and to the liver and eventually expelled the body. The whole process of RCT includes the following steps: the initial peripheral cells contain a large number of cholesterin through the mediating of transporters such as ABCA1 and ABCG1, and then combined with apolipoprotein, HDL, which produces mature state, and then as the blood system aggregated to the liver, and finally excreted the.ATP binding cassette transporter (Binding Cassette Transporter, ABC) belonging to the most present membrane transport family. The ATP tri triphosphate binding cassette transporter A1 (ABCA1) is the most prominent transporter in transport, which helps the cell Cholesterol efflux. Under the action of ABCA1 transporter, lipid may occur Exodus, then combine with apoA-I, and then produce a certain level of phospholipid -apoA-I complex, and can esterification the extracellular free state of cholesterol, and eventually produce a mature HDL, with the blood system aggregation in the liver, and then able to be produced by the liver. Bile salts, and fecal excretion of [1-3]., increase the level of RCT to help prevent AS lesions. Related research results suggest that the number of High Density Lipoprotein Cholesterol, HDL-C is significantly negatively correlated with the possibility of atherosclerosis in the body, and.HDL-C antagonists the main pathogenesis of atherosclerosis The process is mediated by the mediating RCT process, in which RCT can transport a large amount of cholesterol contained in the peripheral tissue through HDL-C to the liver in the form of bile acid, which is currently recognized by researchers in the related fields as a key mechanism for anti atherosclerosis. In RCT, ABCA1 is beneficial to the transport of phosphorus in cells and to the transport of cholesterol to HDL-C precursors apoA-I. New HDL-C, and then start RCT, so ABCA1 plays an extremely important starting speed limit in the new generation of RCT and HDL-C. So can the anti transport of cholesterol be promoted to become a widely concerned target of anti atherosclerosis by regulating the expression of ABCA1 protein. Adiponectin (Adiponectin) is mainly derived from adipocytes and can play the pancreas. Isosin resistance, anti atherosclerotic and anti-inflammatory effects. Studies have shown that there is a lower level of adiponectin in the serum of patients with obesity, hypertension, diabetes, myocardial damage, atherosclerosis and other patients. And many clinical trials show that the concentration of adiponectin is significantly reduced with hypertension, vascular endothelial damage, coronary heart disease and myocardial infarction. The development of the disease is closely related to [4]. in the process of AS disease. Adiponectin can inhibit the differentiation and reproduction of vascular smooth muscle cells, avoid the continuous transformation of macrophages into foam cells, and make adiponectin play the anti AS effect in endothelial cells. The effect of lipoprotein on the reverse transfer of cholesterol in macrophages and its effect on the reverse transport of cholesterol in macrophages and the effect of adiponectin on the reverse transport of cholesterol in macrophages and the effect of adiponectin and its effect on the reverse transport of cholesterol in macrophages and the effect of adiponectin and its effect on the reverse transport of cholesterol in macrophages Mechanism in the early stage of coronary atherosclerotic AS the endothelial cells can be activated by inflammatory cells to be activated on the vascular wall and are dose-dependent inhibition of TNF induced adhesion on the human aortic endothelium. When atherosclerosis or other large vascular lesions occur, a large number of adiponectin will accumulate on the damaged vascular wall and cause the disease. Adiponectin consumes plasma adiponectin with the development of atherosclerosis progressive decline of adiponectin is an independent risk factor for the development of coronary atherosclerosis. Adiponectin is an independent risk factor for the development of coronary atherosclerosis. Sexual adiponectin is often lower than that of women who think that it may be a reason that the incidence of coronary heart disease in women is lower than that of men. The growth of peripheral mononuclear cells and the release of inflammatory factors can protect the endothelium from anti-inflammatory and antagonistic atherosclerosis. Its level is negatively correlated with the triglyceride apolipoprotein and is positively related to high density lipoprotein and apolipoprotein A-I. This topic is to explore the adiponectin to cholesterol through two parts of cell level and animal level. An adiponectin gene knockout mouse (Adiponectin-/-GeneKnockOutMice) was selected as a study animal to keep high fat feeding for 8 weeks and establish atherosclerosis model. The changes of blood lipid levels in adiponectin groups were detected. After 3H-TC injection, the liver and blood of adiponectin treated animal liver and blood were treated with liquid flicker. The amount of cholesterol efflux contained in the feces was detected, and the mRNA level and the protein amount of ABCA1 after adiponectin treatment were detected by Western-blot and PCR. The effect of adiponectin on the production of RCT and the related mechanism were deeply analyzed, so as to provide a new direction for the clinical effective prevention and development of the disease, a new idea of.1, fat. The expression and significance of ABCA1 and ABCG1 in RAW264.7 macrophages was up-regulated through the hepatic X receptor alpha signaling pathway: in vitro whether adiponectin APN affects the ABCA1 in RAW264.7 derived foam cells through the X receptor alpha signaling pathway of the liver, the mRNA level of ABCG1 expression, the amount of protein, the amount of cholesterol efflux, the total cholesterol level. Ox-LDL induced RAW264.7 macrophages were induced as foam cells. The expression of ABCA1, ABCG1mRNA and protein was detected by real-time fluorescence quantitative PCR and Westernblot; the liquid scintillation counting was used to determine the cholesterol efflux rate in lipid THP-1 macrophages and RAW264.7 macrophages. High performance liquid chromatography was used to detect the intracellular cholesterol content. Results: adiponectin. The expression of ABCA1 and ABCG1 in RAW264.7 macrophages was up-regulated through the LXR alpha signaling pathway. Conclusion: Adiponectin enhances the ABCA1, ABCG1 expression and cholesterol efflux in RAW264.7 macrophages through LXR alpha pathway. It provides a theoretical basis for the prevention and treatment of atherosclerosis,.2, ABCA1 expression and atherosclerosis in APN- / - atherosclerotic mice Objective: To study the effect of adiponectin on the reverse transport of cholesterol in macrophages in adiponectin gene knockout mice (APN-/- mice) and the possible mechanism of action in anti atherosclerosis.1. to observe the effect of adiponectin on carotid atherosclerotic plaques in ApoE-/- mice; 2. to explore the effect of adiponectin on the nitrocellulose stress in the plaque. The effect and possible mechanism of the 3. adiponectin on plaque local blood flow shear force and other possible mechanisms to inhibit the plaque. Methods: 1. model establishment: 4 weeks old adiponectin gene knockout mice (Adiponectin-/-geneknockoutmice) 30, high fat feeding for 8 weeks, the establishment of atherosclerotic model.2. grouping: Animal random They were divided into five groups: blank control group, 50 g/ (kg d) intervention group, 150 mu g/ (kg / D) intervention group, 200 g/ (kg. D) intervention group and 250 mu g/ (kg d) intervention group. After 4 weeks of adiponectin intervention, acetylation and 3 macrophage suspension were injected into the abdominal cavity of mice. The serum lipid level of mice was measured by enzyme method, and the liquid flash counting instrument was determined. The percentage of 3H-TC in the liver, serum and feces of mice accounted for the total amount of 3H-TC intraperitoneally injected. RT-PCR and Western-blot were used to detect the mRNA and protein expression levels of ABCA1 in the liver. Results: 1. compared with the control group, the serum TG, TC, LDL level and HDL increased in the adiponectin intervention group, and the changes were dose-dependent with the adiponectin to change the.2. control group. The atherosclerotic lesions were obvious. There was a large amount of lipid deposition in the vascular wall, the formation of atheromatous plaque and the foam cells. The atherosclerotic lesion of the APN-/- mice in the adiponectin intervention group alleviated, and the serum 3 H-TC content of.3. adiponectin was significantly decreased with the dose dependent change of adiponectin (P0.05), the liver and the liver were significantly decreased. 3 H-TC content in the feces was significantly increased (P0.05).4.RT-PCR test results showed that the mRNA transcriptional level of ABCA1 in the liver tissue of the adiponectin intervention group was significantly higher than that of the control group, and the dose dependent increase with adiponectin (P0.05).5.Western-blot results showed that the liver tissue ABCA1 using adiponectin intervention compared with the control group. The level of protein expression increased obviously and increased with adiponectin in a dose-dependent manner (P0.05). Conclusion: 1. adiponectin can reduce the level of serum TC, TG and LDL, increase the level of HDL; 2. adiponectin can reduce the atherosclerosis of aorta in mice; 3. adiponectin can increase the rate of serum cholesterol efflux; 4. adiponectin can increase the liver in a dose dependent manner. The expression of mRNA and protein in the tissue of ABCA1. Adiponectin promotes the reverse transport of cholesterol by up regulation of ABCA1 and promotes intracellular cholesterol efflux, thus delaying the occurrence and development of atherosclerosis and providing a new idea for the prevention and treatment of cardiovascular diseases.

【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R54

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