SIRT1在血管平滑肌源性泡沫细胞迁移中的作用和机制研究

发布时间：2018-05-03 17:58

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》2016年博士论文

【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 cells,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的主要本源是巨噬细胞。与此同时,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。值得关注的是,VSMC源性FC主要位于内皮下而不是VSMC通常所在的中膜。因此,从中膜向内膜的迁移是VSMC摄取中性脂质发生泡沫样变不可缺少的一步。VSMC的迁移先于还是与FC的形成同步发生并不十分清楚。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。本研究的目的就是探究oxLDL诱导VSMC源性FC形成过程中迁移能力的改变以及所涉及的分子机制。去乙酰化酶1 (sirtuinl, 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,PPAR) -γ、PPAR-γ辅助激活因子(PPAR-y coactivator) -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 nitricoxide 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在泡沫化过程中对VSMC迁移的作用却罕有研究报道,因此成为本研究重点探讨的问题。基质金属蛋白酶(matrixmetalloproteinases,MMPs)是锌离子依赖的,可以降解细胞外基质(extracellularmatrix,ECM)中许多组分的蛋白水解酶。MMP-9,也被称为明胶酶B,在AS不稳定斑块中高度表达。MMP-9是VSMC迁移的标志性蛋白。以往的研究证实MMP-9过度表达能够诱使VSMC迁移至血管内膜,而MMP-9-/-小鼠的血管损伤后VSMC迁移和血管损害的程度则明显降低。因此,本研究也检测了 MMP-9的表达以及其潜在调控因子的变化,希望明确VSMC源性FC形成过程中迁移的的分子机制。瞬时受体电位香草醛亚家族 1 (Transient receptor potential vanilloid subfamily member1,TRPV1)已被证实在改善心脑血管疾病(cardiovascular and cerebrovascular diseases,C-CeVD)方面有显著作用。多项研究显示激活TRPV1可通过代谢或自噬等途径减少VSMC内的脂质堆积从而抑制FC的形成。我们前期的研究也证实了激活TRPV1可抑制自发性高血压大鼠轻度炎性的VSMC的增殖。然而,TRPV1在VSMC迁移方面作用的研究很少。本研究中我们探讨了 TRPV1在VSMC源性FC迁移和MMP-9表达方面的作用,并试图了解SIRT1在其中的关键角色,从而希望丰富TRPV1在防治动脉粥硬化方面的保护机制。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(wild type, 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, a-SMA)进行 VSMC 鉴定。2. oxLDL诱导VSMC形成FC,并利用油红O染色和异丙醇萃取法测定细胞内脂质以鉴定FC。3. Transwell实验检测细胞迁移能力。4. SIRT1激活剂SRT1720 (SRT)和拮抗剂尼克酰胺(nicotinamide, Nic)用来调控SIRT1的表达。5. TRPV1激活剂辣椒素(capsaicin,Caps)和拮抗剂辣椒卓平(capsazepin,Capz)用来调控TRPV1的表达。6.蛋白免疫印迹(Westernblot)和免疫荧光分别定量和定性地检测相关蛋白的表达。结果:1. VSMC源性FC的形成伴随着迁移能力和MMP-9蛋白表达的增加。本研究中,油红O染色发现80μg/mL的oxLDL促进了原代培养的VSMC细胞内红色脂滴的堆积,同时细胞内的脂质含量也显著增高,明确了 oxLDL可以诱导VSMC发生泡沫样变而形成FC。Transwell实验结果显示,oxLDL刺激的VSMC与空白对照组细胞相比迁移能力明显增强,而且Western blot结果也表明MMP-9蛋白表达明显升高。2. oxLDL降低了 SIRT1蛋白的表达而促进了 VSMC源性FC的迁移和MMP-9蛋白的高表达。oxLDL刺激VSMC 24小时后SIRT1蛋白的表达明显降低。本实验用SRT和Nic调控SIRT1蛋白的表达,SRT (1μM)可以上调WT-VSMC和oxLDL刺激的VSMC表达 SIRT1,100μM 的 Nic 显著降低了 SRT 上调的 SIRT1。Western blot 和 Transwell实验结果显示相较于单独oxLDL作用,同时予以SRT刺激VSMC后可以上调SIRT1蛋白的表达和下调MMP-9蛋白的表达,而且该效应被SIRT1的拮抗剂Nic所阻断。3. SIRT1可能通过抑制NF-κB信号通路而抑制VSMC源性FC迁移和MMP-9蛋白表达。oxLDL刺激的VSMC表达磷酸化的IκBα (p-IKBα)和核内NF-κB p65蛋白明显增加。相较于单独oxLDL作用,同时予以SRT刺激VSMC后下调了 p-IκBα和核内p65蛋白的表达,而且该效应被SIRT1的拮抗剂Nic所阻断。NF-κB抑制剂BAY 11-7082也显著地降低了 MMP-9蛋白的表达。4. Caps激活TRPV1可抑制VSMC源性FC的迁移和MMP-9蛋白的表达。分别应用TRPV1激动剂Caps和抑制剂Capz来调控TRPV1的表达。Caps呈浓度依赖性地增加TRPV1蛋白的表达而降低MMP-9蛋白的表达。相较于单独oxLDL作用,同时予以Caps刺激VSMC后可以下调MMP-9蛋白的表达和细胞的迁移,而且该效应被TRPV1的拮抗剂Capz所阻断。5. Caps激活TRPV1可上调SIRT1蛋白的表达以及抑制NF-κB信号通路。Caps激活TRPV1呈浓度依赖性地上调VSMC源性FC SIRT1的水平。与对照组相比,Caps刺激VSMC后可以上调SIRT1蛋白的表达,而且该效应被TRPV1的拮抗剂Capz所阻断;Caps刺激VSMC后也可以增加因oxLDL下调的SIRT1蛋白。与单独oxLDL作用相比较,同时予以Caps刺激VSMC后下调了 p-IκBα和核内NF-κB p65蛋白的表达,而且该效应被Capz所阻断。结论:本研究表明oxLDL诱导VSMC源性FC的形成伴随着细胞迁移能力和MMP-9蛋白表达的升高,且证实其与SIRT1的降低和NF-1κB信号通路的增强有关。Caps激活TRPV1后可通过增强SIRT1表达和减弱NF-1κB信号而抑制VSMC源性FC的迁移。泡沫化和表型转化是VSMC参与AS发生发展的关键环节。本研究表明SIRT1是改善VSMC功能的潜在干预靶标,并丰富了 TRPV1在防治AS方面的保护作用。近年来的研究表明白藜芦醇等SIRT1的激活剂已经在AS动物和细胞模型中显示出了保护作用。即使临床结果并不稳定和一致,维持较高水平的SIRT1可能对防治AS有益。
[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 cells (FC).FC forms lipid stripes and even atheromatous plaques is a key pathological link in the early AS. Previous studies have widely believed that the main origin of FC in AS is macrophage. At the same time, a large number of vascular smooth muscle cells (vascular smooth muscle cell) are also stored in AS, especially in the middle and late stages. It is concerned that VSMC derived FC is mainly located under the endothelium rather than the middle membrane usually located in VSMC. Therefore, migration from the middle membrane to the endometrium is an indispensable step for VSMC uptake of neutral lipid foam, or the migration of.VSMC is not ten distinct from the formation of FC. Oxidized low density lipoprotein (oxidized low-densi). Ty lipoprotein, oxLDL) is generally considered to be the strongest AS factor and is proved to promote the foaming of VSMC in vivo and in vitro. The purpose of this study is to explore the changes in migration ability and the molecular mechanism involved in oxLDL induced VSMC derived FC formation. Deacetylase 1 (sirtuinl, SIRT1) is a mammal. The protein that is homologous to the silencing regulator 2 (silent information regulator 2, Sir2), which is a member of the histone deacetylase, can target many downstream proteins and thus affect a variety of pathophysiological processes. These proteins include the peroxidase proliferator activated receptor (peroxisome proliferator-activate). D receptor, PPAR) - gamma, PPAR- gamma auxiliary activating factor (PPAR-y coactivator) -1 a, uncoupling protein (uncoupling protein) -2, liver X receptor (liver) and nuclear factor - kappa, can increase the activity of after acetylation and promote cholesterol to reverse the removal of cholesterol from the cells. Eventually inhibiting the formation of macrophage derived FC.SIRT1 can prevent endothelial dysfunction by inhibiting premature senility and.SIRT1 can also up-regulation the endothelial nitric oxide synthase (endothelial nitricoxide synthase, eNOS) and improve the vasodilatation caused by high fat diet. In addition, it can stabilize plaque, reduce cholesterol intake, and inhibit megagocytosis. The positive effects of cellular foam and alleviating inflammatory reaction make SIRT1 a new target for the prevention and control of AS. However, the role of SIRT1 in the migration of VSMC during the foaming process is rarely reported. Therefore, it has become a key issue in this study. Matrix metalloproteinases (matrixmetalloproteinases, MMPs) are zinc ions dependent and can be reduced. The protein hydrolase.MMP-9 of many components in extracellularmatrix (ECM) is also known as gelatinase B. The high expression of.MMP-9 in AS unstable plaques is a landmark protein for VSMC migration. Previous studies have confirmed that the overexpression of MMP-9 can induce VSMC to migrate to the intima of the blood tube, while the MMP-9-/- mouse's vascular damage is VSMC. The degree of migration and vascular damage was significantly reduced. Therefore, this study also detected the expression of MMP-9 and the changes in its potential regulatory factors, hoping to clarify the molecular mechanism of migration during the formation of VSMC derived FC. The instantaneous receptor potential vanillin subfamily 1 (Transient receptor potential vanilloid subfamily member1, TRPV1) has been proved Cardiovascular and cerebrovascular diseases (C-CeVD) has been significantly improved. A number of studies have shown that activation of TRPV1 can reduce the lipid accumulation in VSMC through metabolic or autophagy and thus inhibit the formation of FC. Our previous study also confirmed that activating TRPV1 can inhibit spontaneous hypertension in rats. VSMC proliferation. However, the role of TRPV1 in VSMC migration is rarely studied. In this study, we explored the role of TRPV1 in VSMC derived FC migration and MMP-9 expression, and tried to understand the key role of SIRT1 in it, so as to enrich the protection mechanism of TRPV1 in the prevention and control of atherosclerosis. Materials and methods: 1. The male C57BL/6J wild type (wild type, WT) mouse thoracic aorta VSMC was cultured by tissue block adhesion method, and the intracellular alpha smooth muscle actin (alpha -smooth muscle actin, a-SMA) was labeled by immunofluorescence and flow cytometry, and VSMC was identified as.2. oxLDL, and the oil red staining and isopropanol extraction were used to determine the VSMC. Determination of intracellular lipid in order to identify the cell migration ability of FC.3. Transwell test,.4. SIRT1 activator SRT1720 (SRT) and antagonist Nik amide (nicotinamide, Nic) used to regulate the expression of SIRT1,.5. TRPV1 activator capsaicin (capsaicin,) and antagonist pepper Zhuo Ping The expression of related proteins was quantitatively and qualitatively detected by Westernblot and immunofluorescence. Results: the formation of 1. VSMC derived FC was accompanied by an increase in mobility and expression of MMP-9 protein. In this study, the oil red O staining found that the oxLDL of 80 mu g/mL promoted the accumulation of red lipid droplets in the primary cultured VSMC cells, and the lipid in the cells. The content was also significantly increased, it was clear that oxLDL could induce VSMC foam change and form FC.Transwell experiment, and the result of FC.Transwell experiment showed that the migration ability of oxLDL stimulated VSMC was obviously enhanced compared with that of blank control group, and Western blot results also showed that the expression of MMP-9 protein was obviously elevated and oxLDL decreased the expression of SIRT1 protein and promoted V. The migration of SMC derived FC and the high expression of MMP-9 protein stimulated VSMC after VSMC for 24 hours. The expression of SIRT1 protein was obviously reduced by SRT and Nic, and SRT (1 mu M) could up regulate the expression of WT-VSMC and stimulating. The results showed that the expression of SIRT1 protein was up-regulated and the expression of MMP-9 protein was down regulated by SRT stimulation of VSMC, and the effect was blocked by SIRT1 antagonist Nic, and.3. SIRT1 could inhibit the VSMC source migration and the expression of phosphorous phosphorous by inhibiting the NF- kappa B signaling pathway. The acidified I kappa B alpha (p-IKB alpha) and the NF- kappa B p65 protein in the nucleus increased significantly. Compared to the single oxLDL action, the expression of p-I kappa B alpha and nuclear p65 protein was down regulated by SRT stimulation at the same time, and the effect was blocked by the antagonist of the antagonist. The expression of the inhibitor 11-7082 also significantly reduced the expression of the protein. Inhibition of the migration of VSMC derived FC and the expression of MMP-9 protein. TRPV1 agonist Caps and inhibitor Capz were used to regulate the expression of TRPV1 in a concentration dependent manner to increase the expression of TRPV1 protein and reduce the expression of MMP-9 protein. Cell migration, and the effect was blocked by the antagonist Capz of TRPV1,.5. Caps activation TRPV1 can up regulate the expression of SIRT1 protein and inhibit NF- kappa B signaling pathway.Caps activation TRPV1 in a concentration dependent dependence on VSMC source FC. The antagonist, Capz, was blocked by the antagonist of PV1; Caps stimulation of VSMC could also increase the SIRT1 protein down regulated by oxLDL. Compared with the single oxLDL action, Caps stimulated VSMC down the expression of p-I kappa B alpha and nuclear NF- kappa protein, and the effect was blocked. The increase in cell migration ability and MMP-9 protein expression, and confirmed that it is related to the decrease of SIRT1 and the enhancement of the NF-1 kappa B signaling pathway, which can inhibit the migration of VSMC derived FC by enhancing the expression of SIRT1 and weakening NF-1 kappa B signal. Foam and phenotypic transformation are the key links to participate in the development of VSMC. It is a potential intervention target for improving the function of VSMC and enriches the protective effect of TRPV1 in the prevention and control of AS. In recent years, it has been shown that the activator of veratrol and other SIRT1 has shown a protective effect in the animal and cell models of AS. Even if the clinical results are not stable and consistent, a higher level of SIRT1 may be beneficial to the prevention and control of AS.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.5

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