本文選題：血管平滑肌細胞 + 氧化型低密度脂蛋白�。� 參考：《第三軍醫(yī)大學(xué)》2016年博士論文

【摘要】：背景和目的:動脈粥樣硬化(atherosclerosis,AS)因可引起缺血性心臟病、腦卒中和外周血管疾病等高發(fā)病率和高致死率的疾病而成為全世界范圍內(nèi)人口死亡的首要原因。膽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細胞內(nèi)形成泡沫細胞(foam cells,FC)。FC形成脂質(zhì)條紋以至粥樣斑塊是AS早期的關(guān)鍵病理環(huán)節(jié)。既往研究廣泛認為AS中FC的主要本源是巨噬細胞。與此同時,AS中還大量存著血管平滑肌細胞(vascular smooth muscle cell,VSMC)來源的FC,中晚期病變中尤為顯著。值得關(guān)注的是,VSMC源性FC主要位于內(nèi)皮下而不是VSMC通常所在的中膜。因此,從中膜向內(nèi)膜的遷移是VSMC攝取中性脂質(zhì)發(fā)生泡沫樣變不可缺少的一步。VSMC的遷移先于還是與FC的形成同步發(fā)生并不十分清楚。氧化型低密度脂蛋白(oxidized low-density lipoprotein, oxLDL) 通常認為是最強的致 AS 因子,且在體內(nèi)體外研究中皆證實可以促進VSMC發(fā)生泡沫化。本研究的目的就是探究oxLDL誘導(dǎo)VSMC源性FC形成過程中遷移能力的改變以及所涉及的分子機制。去乙�；�1 (sirtuinl, SIRT1)是哺乳動物內(nèi)與酵母菌的沉默信息調(diào)控因子2(silent information regulator 2,Sir2)同源的蛋白,是組蛋白去乙�；赋蓡T之一。SIRT1可以靶定許多下游蛋白從而影響多種病理生理過程,這些蛋白包括:過氧化物酶體增殖物活化受體(peroxisome proliferator-activated receptor,PPAR) -γ、PPAR-γ輔助激活因子(PPAR-y coactivator) -1α、解偶聯(lián)蛋白(uncoupling protein) -2、肝 X受體(liver X receptor,LXR)和核因子(nuclear factor,NF) -κB 等。SIRT1 使 LXR去乙�；罂梢陨险{(diào)后者的活性,并促進膽固醇逆轉(zhuǎn)運將膽固醇從細胞內(nèi)排出,最終抑制巨噬細胞源性FC的形成。SIRT1可以通過抑制早衰而防止內(nèi)皮功能紊亂。SIRT1還可以上調(diào)內(nèi)皮型一氧化氮合酶(endothelial nitricoxide synthase,eNOS)而改善因高脂飲食受損的血管舒張功能。此外,在穩(wěn)定斑塊、降低膽固醇攝取、抑制巨噬細胞泡沫化以及減輕炎癥反應(yīng)等方面的積極作用使SIRT1成為防治AS的新靶標。然而SIRT1在泡沫化過程中對VSMC遷移的作用卻罕有研究報道,因此成為本研究重點探討的問題。基質(zhì)金屬蛋白酶(matrixmetalloproteinases,MMPs)是鋅離子依賴的,可以降解細胞外基質(zhì)(extracellularmatrix,ECM)中許多組分的蛋白水解酶。MMP-9,也被稱為明膠酶B,在AS不穩(wěn)定斑塊中高度表達。MMP-9是VSMC遷移的標志性蛋白。以往的研究證實MMP-9過度表達能夠誘使VSMC遷移至血管內(nèi)膜,而MMP-9-/-小鼠的血管損傷后VSMC遷移和血管損害的程度則明顯降低。因此,本研究也檢測了 MMP-9的表達以及其潛在調(diào)控因子的變化,希望明確VSMC源性FC形成過程中遷移的的分子機制。瞬時受體電位香草醛亞家族 1 (Transient receptor potential vanilloid subfamily member1,TRPV1)已被證實在改善心腦血管疾病(cardiovascular and cerebrovascular diseases,C-CeVD)方面有顯著作用。多項研究顯示激活TRPV1可通過代謝或自噬等途徑減少VSMC內(nèi)的脂質(zhì)堆積從而抑制FC的形成。我們前期的研究也證實了激活TRPV1可抑制自發(fā)性高血壓大鼠輕度炎性的VSMC的增殖。然而,TRPV1在VSMC遷移方面作用的研究很少。本研究中我們探討了 TRPV1在VSMC源性FC遷移和MMP-9表達方面的作用,并試圖了解SIRT1在其中的關(guān)鍵角色,從而希望豐富TRPV1在防治動脈粥硬化方面的保護機制。材料與方法:1.采用組織塊貼壁法原代培養(yǎng)雄性C57BL/6J野生型(wild type, WT)小鼠胸主動脈VSMC,并利用免疫熒光和流式細胞技術(shù)標記細胞內(nèi)α平滑肌肌動蛋白(α-smooth muscle actin, a-SMA)進行 VSMC 鑒定。2. oxLDL誘導(dǎo)VSMC形成FC,并利用油紅O染色和異丙醇萃取法測定細胞內(nèi)脂質(zhì)以鑒定FC。3. Transwell實驗檢測細胞遷移能力。4. SIRT1激活劑SRT1720 (SRT)和拮抗劑尼克酰胺(nicotinamide, Nic)用來調(diào)控SIRT1的表達。5. TRPV1激活劑辣椒素(capsaicin,Caps)和拮抗劑辣椒卓平(capsazepin,Capz)用來調(diào)控TRPV1的表達。6.蛋白免疫印跡(Westernblot)和免疫熒光分別定量和定性地檢測相關(guān)蛋白的表達。結(jié)果:1. VSMC源性FC的形成伴隨著遷移能力和MMP-9蛋白表達的增加。本研究中,油紅O染色發(fā)現(xiàn)80μg/mL的oxLDL促進了原代培養(yǎng)的VSMC細胞內(nèi)紅色脂滴的堆積,同時細胞內(nèi)的脂質(zhì)含量也顯著增高,明確了 oxLDL可以誘導(dǎo)VSMC發(fā)生泡沫樣變而形成FC。Transwell實驗結(jié)果顯示,oxLDL刺激的VSMC與空白對照組細胞相比遷移能力明顯增強,而且Western blot結(jié)果也表明MMP-9蛋白表達明顯升高。2. oxLDL降低了 SIRT1蛋白的表達而促進了 VSMC源性FC的遷移和MMP-9蛋白的高表達。oxLDL刺激VSMC 24小時后SIRT1蛋白的表達明顯降低。本實驗用SRT和Nic調(diào)控SIRT1蛋白的表達,SRT (1μM)可以上調(diào)WT-VSMC和oxLDL刺激的VSMC表達 SIRT1,100μM 的 Nic 顯著降低了 SRT 上調(diào)的 SIRT1。Western blot 和 Transwell實驗結(jié)果顯示相較于單獨oxLDL作用,同時予以SRT刺激VSMC后可以上調(diào)SIRT1蛋白的表達和下調(diào)MMP-9蛋白的表達,而且該效應(yīng)被SIRT1的拮抗劑Nic所阻斷。3. SIRT1可能通過抑制NF-κB信號通路而抑制VSMC源性FC遷移和MMP-9蛋白表達。oxLDL刺激的VSMC表達磷酸化的IκBα (p-IKBα)和核內(nèi)NF-κB p65蛋白明顯增加。相較于單獨oxLDL作用,同時予以SRT刺激VSMC后下調(diào)了 p-IκBα和核內(nèi)p65蛋白的表達,而且該效應(yīng)被SIRT1的拮抗劑Nic所阻斷。NF-κB抑制劑BAY 11-7082也顯著地降低了 MMP-9蛋白的表達。4. Caps激活TRPV1可抑制VSMC源性FC的遷移和MMP-9蛋白的表達。分別應(yīng)用TRPV1激動劑Caps和抑制劑Capz來調(diào)控TRPV1的表達。Caps呈濃度依賴性地增加TRPV1蛋白的表達而降低MMP-9蛋白的表達。相較于單獨oxLDL作用,同時予以Caps刺激VSMC后可以下調(diào)MMP-9蛋白的表達和細胞的遷移,而且該效應(yīng)被TRPV1的拮抗劑Capz所阻斷。5. Caps激活TRPV1可上調(diào)SIRT1蛋白的表達以及抑制NF-κB信號通路。Caps激活TRPV1呈濃度依賴性地上調(diào)VSMC源性FC SIRT1的水平。與對照組相比,Caps刺激VSMC后可以上調(diào)SIRT1蛋白的表達,而且該效應(yīng)被TRPV1的拮抗劑Capz所阻斷;Caps刺激VSMC后也可以增加因oxLDL下調(diào)的SIRT1蛋白。與單獨oxLDL作用相比較,同時予以Caps刺激VSMC后下調(diào)了 p-IκBα和核內(nèi)NF-κB p65蛋白的表達,而且該效應(yīng)被Capz所阻斷。結(jié)論:本研究表明oxLDL誘導(dǎo)VSMC源性FC的形成伴隨著細胞遷移能力和MMP-9蛋白表達的升高,且證實其與SIRT1的降低和NF-1κB信號通路的增強有關(guān)。Caps激活TRPV1后可通過增強SIRT1表達和減弱NF-1κB信號而抑制VSMC源性FC的遷移。泡沫化和表型轉(zhuǎn)化是VSMC參與AS發(fā)生發(fā)展的關(guān)鍵環(huán)節(jié)。本研究表明SIRT1是改善VSMC功能的潛在干預(yù)靶標,并豐富了 TRPV1在防治AS方面的保護作用。近年來的研究表明白藜蘆醇等SIRT1的激活劑已經(jīng)在AS動物和細胞模型中顯示出了保護作用。即使臨床結(jié)果并不穩(wěn)定和一致,維持較高水平的SIRT1可能對防治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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R543.5

【參考文獻】

相關(guān)期刊論文 前3條

1 陳偉偉;高潤霖;劉力生;朱曼璐;王文;王擁軍;吳兆蘇;李惠君;鄭哲;蔣立新;胡盛壽;;《中國心血管病報告2014》概要[J];中國循環(huán)雜志;2015年07期

2 劉峗;李敬誠;尹延偉;黎炳護;郭露;廖少瓊;曹小潔;張明杰;周毅;陳磊;龍春燕;張莉莉;;激活TRPV1對自發(fā)性高血壓大鼠血管平滑肌細胞增殖的影響[J];第三軍醫(yī)大學(xué)學(xué)報;2015年08期

3 Li Li;Peng Gao;Hou-zao Chen;Zhu-qin Zhang;Ting-ting Xu;Yu-yan Jia;Hui-na Zhang;Guan-hua Du;De-pei Liu;;Up-regulation of Fas Ligand Expression by Sirtuin 1 in both Flow-restricted Vessels and Serum-stimulated Vascular Smooth Muscle Cells[J];Chinese Medical Sciences Journal;2013年02期

相關(guān)博士學(xué)位論文 前1條

1 張慧娜;Sirt1對血管平滑肌細胞遷移的影響及分子機制研究[D];中國協(xié)和醫(yī)科大學(xué);2008年

，

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