oxLDL诱导血管平滑肌泡沫细胞形成及其机制的研究

发布时间：2018-04-22 10:07

本文选题：血管平滑肌细胞 + 氧化型低密度脂蛋白　；参考：《第三军医大学》2017年硕士论文

【摘要】：背景和目的:动脉粥样硬化(atherosclerosis,AS)因可引起缺血性心脏病、脑卒中和外周血管疾病等高发病率和高致死率的疾病而成为全世界范围内人口死亡的首要原因。胆固醇酯和甘油三酯等中性脂肪以脂滴的形式存在于血管壁细胞内形成泡沫细胞(foam cell,FC)。FC形成脂质条纹以至粥样斑块是AS早期的关键病理环节。既往研究广泛认为AS中FC的主要来源于巨噬细胞。近年的研究认识到,AS中还大量存着血管平滑肌细胞(vascular smooth muscle cell,VSMC)来源的FC,中晚期病变中尤为显著。氧化型低密度脂蛋白(oxidized low-density lipoprotein,oxLDL)通常认为是最强的致AS因子,且在体内体外研究中皆证实可以促进VSMC发生泡沫化。传统的观点认为oxLDL主要在细胞膜上清道夫受体的介导下进入细胞内。但是越来越多的研究发现,各种致动脉硬化因子可促进VSMC的单核巨噬细胞样转化,从而使得VSMC细胞具备了一定的胞饮功能。本研究的目的就是探究oxLDL诱导VSMC源性FC形成过程中大胞饮在摄取脂质的作用以及SIRT1的作用。大胞饮(macropinocytosis)已被证实在巨噬细胞源性FC形成过程中起到重要作用。多项研究证实巨噬细胞可以通过非选择性大胞饮的形式大量摄取细胞外的修饰或非修饰LDL,并且形成泡沫细胞。但是在VSMC FC形成过程中,是否也有大胞饮途径的参与,具体又是通过怎样的机制,目前还未见研究报道。之前有报道证实,PDGF可以刺激VSMC以大胞饮方式摄取药物颗粒,同时VSMC可以以大胞饮形式摄取小鼠血清中的LDL形成泡沫细胞,因此本研究中应用鼠源性PDGF-BB刺激VSMC,并探讨是否会出现大胞饮以及oxLDL能否通过这种形式进入细胞内。去乙酰化酶1(sirtuin1,SIRT1)是哺乳动物内与酵母菌的沉默信息调控因子2(silent information regulator 2,Sir2)同源的蛋白,是组蛋白去乙酰化酶成员之一。SIRT1可以靶定许多下游蛋白从而影响多种病理生理过程,这些蛋白包括:过氧化物酶体增殖物活化受体(peroxisome proliferator-activated receptor,PPARγ)、PPARγ辅助激活因子(PPARγcoactivator-1α)、解偶联蛋白(uncoupling protein-2)、肝X受体(liver X receptor,LXR)和核因子(nuclear factor,NF-κB)等。SIRT1使LXR去乙酰化后可以上调后者的活性,并促进胆固醇逆转运将胆固醇从细胞内排出,最终抑制巨噬细胞源性FC的形成。SIRT1可以通过抑制早衰而防止内皮功能紊乱。SIRT1还可以上调内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)而改善因高脂饮食受损的血管舒张功能。此外,在稳定斑块、降低胆固醇摄取、抑制巨噬细胞泡沫化以及减轻炎症反应等方面的积极作用使SIRT1成为防治AS的新靶标。然而SIRT1在VSMC泡沫化过程中能否起到作用却罕有研究报道,因此本研究重点探讨SIRT1在VSMC泡沫化中的作用及其相应机制。酰基辅酶A:胆固醇酰基转移酶1(A-cholesterol acyltransferase 1,ACAT1)是细胞内催化游离胆固醇和脂肪酸合成胆固醇酯,进而形成泡沫细胞的关键酶。以往研究证实ACAT1在巨噬细胞和VSMC泡沫化胆固醇酯合成中起到关键作用,那么SIRT1是否可以影响ACAT1表达来干预VSMC泡沫化,本研究将检测FC形成过程中ACAT1的表达情况来明确其作用。之前已有报道SIRT1可以与PPARγ直接作用,同时在巨噬细胞和VSMC中PPARγ可以直接调控ACAT1作用,本实验中我们同时检测了PPARγ表达以明确其在FC形成过程中的具体作用和机制。材料与方法:1.采用组织块贴壁法原代培养雄性C57BL/6J野生型(wildtype,WT)小鼠胸主动脉VSMC,并利用免疫荧光和流式细胞技术标记细胞内α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)进行VSMC鉴定。2.oxLDL诱导VSMC形成FC,并利用油红O染色鉴定FC。3.PDGF-BB刺激VSMC,使用胞饮示踪剂Lucifer Yellow(LY)以及荧光标记的LDL和oxLDL(Di I-LDL和Di I-oxLDL),应用流式细胞术检测细胞荧光。4.PDGF-BB刺激VSMC,使用肌动蛋白聚合抑制剂细胞松弛素D(Cytochalasin D,CyD),PI3-K抑制剂LY 294002,Na+/H+交换抑制剂阿米洛利(Amiloride),动力素抑制剂Dynasore,网格蛋白依赖内吞抑制剂高渗蔗糖(hypertonic sucrose),用流式细胞术观察PDGF刺激下VSMC对oxLDL摄取情况的影响。5.SIRT1激活剂SRT1720(SRT)和拮抗剂尼克酰胺(nicotinamide,Nic)用来调控SIRT1的表达。6.PPARγ激活剂Rosiglitazone(RSG)和抑制剂GW9662调控PPARγ蛋白表达。7.蛋白免疫印迹(Western blot)定量性地检测相关蛋白的表达;油红O染色检测SRIT1对FC形成的影响。结果:1.PDGF促进VSMC对LDL和oxLDL的摄取,同时胞饮作用增强。PDGF刺激的VSMC可以显著增加含有LY的细胞百分比。随着PDGF浓度梯度增加,可以增加VSMC对LDL的摄取。相似的,使用PDGF可以刺激VSMC增加对oxLDL的摄取。2.大胞饮抑制剂和受体途径抑制剂均可抑制VSMC对oxLDL的摄取使用肌动蛋白聚合抑制剂细胞松弛素D(Cytochalasin D,CyD),PI3-K抑制剂LY 294002可以显著抑制VSMC对oxLDL的摄取;而动力素抑制剂Dynasore也可以显著抑制VSMC对oxLDL的摄取。3.SIRT1抑制ox-LDL诱导的VSMC泡沫细胞形成本研究中,80μg/m L oxLDL刺激VSMC 48小时后,油红O染色发现的oxLDL促进了原代培养的VSMC细胞内红色脂滴的堆积,明确了oxLDL可以诱导VSMC发生泡沫样变而形成FC;同时予以SRT刺激,可以显著减少泡沫细胞,细胞内红色脂滴显著减少;而这种效应被SIRT1的拮抗剂Nic所阻断。Western blot结果证明oxLDL刺激VSMC 24小时后其中SIRT1蛋白表达显著降低。4.SIRT1通过降低ACAT1并升高PPARγ表达,从而抑制FC形成。oxLDL刺激VSMC 24小时后ACAT1蛋白的表达明显上升伴随着PPARγ的下降。本实验用SRT和Nic调控SIRT1蛋白的表达,SRT可以下调ACAT1同时上调PPARγ的表达,Nic则逆转由SRT引发ACAT1和PPARγ的表达。下一步应用PPARγ激活剂Rosiglitazone(RSG)和抑制剂GW9662调控PPARγ蛋白表达,RSG可以显著抑制ACAT1的表达,而GW9662可以逆转这种作用。结论:本研究表明PDGF可以显著促进VSMC对oxLDL的摄取,大胞饮途径介导了VSMC对oxLDL的摄取。SIRT1可以抑制oxLDL诱导VSMC源性FC形成,oxLDL诱导VSMC泡沫样变过程中SIRT1的表达降低。激活SIRT1可抑制VSMC源性FC形成,伴随着ACAT1表达的降低,同时PPARγ表达增高。本研究表明大胞饮途径参与了PDGF诱导的VSMC对oxLDL的摄取;SIRT1可抑制VSMC对oxLDL的摄取和FC的形成,从而起到抑制动脉粥样硬化病变的重要作用。
[Abstract]:Background and purpose: atherosclerosis (AS) is the leading cause of death in the world because of the high incidence of ischemic heart disease, cerebral apoplexy, and peripheral vascular disease and high mortality. The neutral fat, such as cholesterol ester and triglyceride, exists in the form of blood vessel wall form in the form of lipid droplets. Foam cell (FC).FC forms lipid stripes and even atheromatous plaques is a key pathological link in early AS. Previous studies widely believed that FC was mainly derived from macrophages in AS. In recent years, a large number of vascular smooth muscle cells (vascular smooth muscle cell) were found in AS, especially in the middle and late stages. Significant. Oxidized low-density lipoprotein (oxLDL) is generally considered to be the strongest AS factor and is proved to promote the foaming of VSMC in both in vivo and in vitro studies. The traditional view is that oxLDL is mainly mediated by the cell membrane on the membrane of the scavenger receptor. But more and more studies have been made. At present, a variety of arteriosclerosis factors can promote the monocyte macrophage like transformation of VSMC, which makes VSMC cells have a certain function of drinking. The purpose of this study is to explore the effect of oxLDL on the uptake of lipid in the formation of VSMC derived FC and the effect of SIRT1. The large cell drink (macropinocytosis) has been proved to be in macroophagy. A number of studies have shown that macrophages can absorb a large amount of extracellular modified or non modified LDL through non selective large cell drink and form foam cells. But in the process of the formation of VSMC FC, there is also the participation of large cell drink pathway, and what mechanism is through the mechanism. It has not been reported before. Previously, it was reported that PDGF could stimulate VSMC to take drug particles in the way of large cell drink, while VSMC can take the LDL form foam cells in the mouse serum in the form of large cell drink. Therefore, this study used mouse derived PDGF-BB to stimulate VSMC, and whether there would be large cell drink and whether oxLDL could pass this way. Form into the cell. Deacetylase 1 (sirtuin1, SIRT1) is the homologous protein of the silence information regulator 2 (silent information regulator 2, Sir2) in mammalian and yeast. It is one of the members of the histone deacetylase that can target many downstream proteins and thus affect a variety of pathophysiological processes. These proteins include: Peroxisome proliferator-activated receptor, PPAR gamma, PPAR gamma auxiliary activator (PPAR gamma coactivator-1 alpha), uncoupling protein (uncoupling protein-2), liver X receptor (liver X) and nuclear factor, can increase the activity of the latter after deacetylation. It also promotes cholesterol reversal transport of cholesterol from the cells and eventually inhibits the formation of macrophage derived FC.SIRT1, which can prevent endothelial dysfunction by inhibiting premature aging and.SIRT1 can also up regulate the endothelial nitric oxide synthase (endothelial nitric oxide synthase, eNOS) and improve the vasodilatation caused by the hyperlipidemic diet. In addition, the positive effects of stabilizing the plaque, reducing the uptake of cholesterol, inhibiting the foaming of macrophages and reducing the inflammatory reaction make SIRT1 a new target for the prevention and control of AS. However, it is rare to study whether SIRT1 can play a role in the process of VSMC foam. Therefore, this study focuses on the role and phase of SIRT1 in the foaming of VSMC. Acyl coenzyme A: cholesterol acyl transferase 1 (A-cholesterol acyltransferase 1, ACAT1) is a key enzyme that catalyzes the synthesis of cholesteryl esters from intracellular free cholesterol and fatty acids in cells, and then forms a key enzyme in foam cells. Previous studies have confirmed that ACAT1 plays a key role in the synthesis of macrophages and VSMC foamed bile esters. Then SIRT1 is the key factor. Whether or not the expression of ACAT1 can interfere with VSMC foam, this study will determine the expression of ACAT1 during the formation of FC to clarify its role. Previously, it has been reported that SIRT1 can act directly with PPAR gamma, while PPAR gamma can directly regulate the effect of ACAT1 in macrophages and VSMC. In this case, we also detected the expression of PPAR gamma to clear it. The specific functions and mechanisms in the process of FC formation. Materials and methods: 1. the male C57BL/6J wild type (wildtype, WT) mouse thoracic aorta VSMC was cultured by tissue block adhesion method, and the intracellular alpha smooth muscle actin (alpha -smooth muscle actin, alpha -SMA) was labeled by immunofluorescence and flow cytometry for.2.oxLDL induction of VSMC. VSMC forms FC and uses oil red O staining to identify FC.3.PDGF-BB stimulation VSMC, Lucifer Yellow (LY) and LDL and oxLDL (Di I-LDL and oxLDL), which are labeled by fluorescently, and use flow cytometry to detect cell fluorescence stimulation, and use actin polymerization inhibitor cytochalasin. Preparation LY 294002, Na+/H+ exchange inhibitor amiloride (Amiloride), power hormone inhibitor Dynasore, grid protein dependent endocytic inhibitor hypertonic sucrose (hypertonic sucrose), the effect of VSMC on oxLDL uptake by PDGF stimulated by flow cytometry,.5.SIRT1 activator SRT1720 (SRT) and antagonist Nik amide To regulate the expression of.6.PPAR gamma activator Rosiglitazone (RSG) and inhibitor GW9662 to regulate the expression of PPAR gamma protein to express the expression of.7. protein (Western blot) in quantitative detection of the expression of related proteins; oil red O staining was used to detect the effect of SRIT1 on FC formation. Stimulated VSMC can significantly increase the percentage of cells containing LY. With the increase of PDGF concentration gradient, VSMC can increase the uptake of LDL. Similar, PDGF can stimulate VSMC to increase the uptake of oxLDL,.2. large cell drink inhibitors and receptor pathway inhibitors can inhibit VSMC to oxLDL uptake of actin polymerization inhibitors cell pine Relaxin D (Cytochalasin D, CyD), PI3-K inhibitor LY 294002 can significantly inhibit the uptake of VSMC to oxLDL, while the power factor inhibitor Dynasore also significantly inhibits the shape cost of VSMC against oxLDL uptake induced by.3.SIRT1 inhibition. 80 The accumulation of red lipid droplets in the primary culture of VSMC cells showed that oxLDL could induce VSMC to induce foam change and form FC. At the same time, SRT stimulation could significantly reduce the foam cells and the red lipid droplets in the cells decreased significantly; this effect was proved to be 24 hours after oxLDL stimulation VSMC by SIRT1 antagonist Nic. The expression of SIRT1 protein significantly reduced the expression of.4.SIRT1 by reducing ACAT1 and increasing the expression of PPAR gamma, thus inhibiting the FC formation of.OxLDL to stimulate VSMC 24 hours after VSMC, and the expression of ACAT1 protein was obviously increased with the decrease of PPAR gamma. RT induces the expression of ACAT1 and PPAR gamma. The next step is to use PPAR gamma activator Rosiglitazone (RSG) and inhibitor GW9662 to regulate the expression of PPAR gamma protein. RSG can significantly inhibit the expression of ACAT1, while GW9662 can reverse this effect. Conclusion: This study showed that PDGF can significantly promote the uptake of ACAT1. The uptake of.SIRT1 could inhibit oxLDL induced VSMC derived FC formation, and oxLDL induced the decrease of SIRT1 expression during VSMC foam transformation. Activated SIRT1 inhibited the formation of VSMC derived FC, accompanied by a decrease in ACAT1 expression, and increased the expression of PPAR gamma. The uptake of oxLDL and the formation of FC play an important role in inhibiting atherosclerotic lesions.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

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