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》2017年碩士論文

【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 cell,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的主要來源于巨噬細胞。近年的研究認識到,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。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。傳統(tǒng)的觀點認為oxLDL主要在細胞膜上清道夫受體的介導下進入細胞內(nèi)。但是越來越多的研究發(fā)現(xiàn),各種致動脈硬化因子可促進VSMC的單核巨噬細胞樣轉(zhuǎn)化,從而使得VSMC細胞具備了一定的胞飲功能。本研究的目的就是探究oxLDL誘導VSMC源性FC形成過程中大胞飲在攝取脂質(zhì)的作用以及SIRT1的作用。大胞飲(macropinocytosis)已被證實在巨噬細胞源性FC形成過程中起到重要作用。多項研究證實巨噬細胞可以通過非選擇性大胞飲的形式大量攝取細胞外的修飾或非修飾LDL,并且形成泡沫細胞。但是在VSMC FC形成過程中,是否也有大胞飲途徑的參與,具體又是通過怎樣的機制,目前還未見研究報道。之前有報道證實,PDGF可以刺激VSMC以大胞飲方式攝取藥物顆粒,同時VSMC可以以大胞飲形式攝取小鼠血清中的LDL形成泡沫細胞,因此本研究中應用鼠源性PDGF-BB刺激VSMC,并探討是否會出現(xiàn)大胞飲以及oxLDL能否通過這種形式進入細胞內(nèi)。去乙�；�1(sirtuin1,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,PPARγ)、PPARγ輔助激活因子(PPARγcoactivator-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 nitric oxide 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等方面的積極作用使SIRT1成為防治AS的新靶標。然而SIRT1在VSMC泡沫化過程中能否起到作用卻罕有研究報道,因此本研究重點探討SIRT1在VSMC泡沫化中的作用及其相應機制。�；o酶A:膽固醇�；D(zhuǎn)移酶1(A-cholesterol acyltransferase 1,ACAT1)是細胞內(nèi)催化游離膽固醇和脂肪酸合成膽固醇酯,進而形成泡沫細胞的關(guān)鍵酶。以往研究證實ACAT1在巨噬細胞和VSMC泡沫化膽固醇酯合成中起到關(guān)鍵作用,那么SIRT1是否可以影響ACAT1表達來干預VSMC泡沫化,本研究將檢測FC形成過程中ACAT1的表達情況來明確其作用。之前已有報道SIRT1可以與PPARγ直接作用,同時在巨噬細胞和VSMC中PPARγ可以直接調(diào)控ACAT1作用,本實驗中我們同時檢測了PPARγ表達以明確其在FC形成過程中的具體作用和機制。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(wildtype,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,α-SMA)進行VSMC鑒定。2.oxLDL誘導VSMC形成FC,并利用油紅O染色鑒定FC。3.PDGF-BB刺激VSMC,使用胞飲示蹤劑Lucifer Yellow(LY)以及熒光標記的LDL和oxLDL(Di I-LDL和Di I-oxLDL),應用流式細胞術(shù)檢測細胞熒光。4.PDGF-BB刺激VSMC,使用肌動蛋白聚合抑制劑細胞松弛素D(Cytochalasin D,CyD),PI3-K抑制劑LY 294002,Na+/H+交換抑制劑阿米洛利(Amiloride),動力素抑制劑Dynasore,網(wǎng)格蛋白依賴內(nèi)吞抑制劑高滲蔗糖(hypertonic sucrose),用流式細胞術(shù)觀察PDGF刺激下VSMC對oxLDL攝取情況的影響。5.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。6.PPARγ激活劑Rosiglitazone(RSG)和抑制劑GW9662調(diào)控PPARγ蛋白表達。7.蛋白免疫印跡(Western blot)定量性地檢測相關(guān)蛋白的表達;油紅O染色檢測SRIT1對FC形成的影響。結(jié)果:1.PDGF促進VSMC對LDL和oxLDL的攝取,同時胞飲作用增強。PDGF刺激的VSMC可以顯著增加含有LY的細胞百分比。隨著PDGF濃度梯度增加,可以增加VSMC對LDL的攝取。相似的,使用PDGF可以刺激VSMC增加對oxLDL的攝取。2.大胞飲抑制劑和受體途徑抑制劑均可抑制VSMC對oxLDL的攝取使用肌動蛋白聚合抑制劑細胞松弛素D(Cytochalasin D,CyD),PI3-K抑制劑LY 294002可以顯著抑制VSMC對oxLDL的攝取;而動力素抑制劑Dynasore也可以顯著抑制VSMC對oxLDL的攝取。3.SIRT1抑制ox-LDL誘導的VSMC泡沫細胞形成本研究中,80μg/m L oxLDL刺激VSMC 48小時后,油紅O染色發(fā)現(xiàn)的oxLDL促進了原代培養(yǎng)的VSMC細胞內(nèi)紅色脂滴的堆積,明確了oxLDL可以誘導VSMC發(fā)生泡沫樣變而形成FC;同時予以SRT刺激,可以顯著減少泡沫細胞,細胞內(nèi)紅色脂滴顯著減少;而這種效應被SIRT1的拮抗劑Nic所阻斷。Western blot結(jié)果證明oxLDL刺激VSMC 24小時后其中SIRT1蛋白表達顯著降低。4.SIRT1通過降低ACAT1并升高PPARγ表達,從而抑制FC形成。oxLDL刺激VSMC 24小時后ACAT1蛋白的表達明顯上升伴隨著PPARγ的下降。本實驗用SRT和Nic調(diào)控SIRT1蛋白的表達,SRT可以下調(diào)ACAT1同時上調(diào)PPARγ的表達,Nic則逆轉(zhuǎn)由SRT引發(fā)ACAT1和PPARγ的表達。下一步應用PPARγ激活劑Rosiglitazone(RSG)和抑制劑GW9662調(diào)控PPARγ蛋白表達,RSG可以顯著抑制ACAT1的表達,而GW9662可以逆轉(zhuǎn)這種作用。結(jié)論:本研究表明PDGF可以顯著促進VSMC對oxLDL的攝取,大胞飲途徑介導了VSMC對oxLDL的攝取。SIRT1可以抑制oxLDL誘導VSMC源性FC形成,oxLDL誘導VSMC泡沫樣變過程中SIRT1的表達降低。激活SIRT1可抑制VSMC源性FC形成,伴隨著ACAT1表達的降低,同時PPARγ表達增高。本研究表明大胞飲途徑參與了PDGF誘導的VSMC對oxLDL的攝取;SIRT1可抑制VSMC對oxLDL的攝取和FC的形成,從而起到抑制動脈粥樣硬化病變的重要作用。
[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.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R543.5

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相關(guān)期刊論文 前1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;顧東風;楊躍進;鄭哲;蔣立新;胡盛壽;;《中國心血管病報告2015》概要[J];中國循環(huán)雜志;2016年06期

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