本文關(guān)鍵詞： 動脈粥樣硬化 易損斑塊 脂質(zhì)代謝 人參皂苷Rb1 自噬　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景動脈粥樣硬化(atherosclerosis,AS)斑塊破裂和血栓形成是導(dǎo)致急性心腦血管事件的主要病理機(jī)制,對可能發(fā)生破裂的易損斑塊(vulnerable plaque)進(jìn)行早期積極干預(yù)是預(yù)防和減低急性心腦血管事件的發(fā)生率的重要方法。AS是一種脂質(zhì)驅(qū)動的慢性炎癥性疾病。其主要病理表現(xiàn)為脂質(zhì)堆積和炎癥細(xì)胞浸潤,巨噬細(xì)胞在這一過程中發(fā)揮重要作用。血液中的低密度脂蛋白膽固醇被巨噬細(xì)胞吞噬后以膽固醇酯的形式存在于脂滴中,蓄積了大量脂質(zhì)的巨噬細(xì)胞變?yōu)榕菽?xì)胞。如何減少泡沫細(xì)胞中脂質(zhì)的蓄積,對于降低斑塊脂負(fù)荷、促進(jìn)斑塊穩(wěn)定具有重要意義。人參皂苷Rb1是人參當(dāng)中含量最多的活性成分。目前的研究發(fā)現(xiàn),Rb1具有降低血脂、降低體重、對抗炎癥、抑制血管形成和增強(qiáng)胰島素敏感性等生理作用。但是,Rb1在動脈粥樣硬化中的作用尚沒有研究。本課題擬通過構(gòu)建AS小鼠模型和體外培養(yǎng)巨噬細(xì)胞,探討人參皂苷Rb1通過調(diào)控巨噬細(xì)胞自噬對動脈粥樣硬化斑塊穩(wěn)定性的作用及其相關(guān)機(jī)制。目的通過構(gòu)建體外泡沫細(xì)胞模型及動脈粥樣硬化小鼠易損斑塊模型,研究人參皂苷Rb1調(diào)控巨噬細(xì)胞自噬、影響脂質(zhì)代謝及穩(wěn)定AS易損斑塊的分子機(jī)制。方法在體外實驗中,我們提取C57BL/6小鼠腹腔原代巨噬細(xì)胞,給予ox-LDL(100μg/ml)刺激24小時誘導(dǎo)成泡沫細(xì)胞,然后分別給予不同濃度Rb1處理24小時。油紅O染色檢測各組細(xì)胞脂質(zhì)沉積情況,western blot檢測自噬蛋白LC3Ⅱ和SQSTM1含量,電鏡觀察細(xì)胞中脂滴及自噬體膜形成數(shù)量;通過慢病毒轉(zhuǎn)染Atg5 siRNA抑制細(xì)胞自噬過程,觀察干預(yù)自噬前后,Rb1對巨噬細(xì)胞脂質(zhì)代謝的影響;給予AMPK抑制劑Compond C,研究AMPK磷酸化在Rb1介導(dǎo)的自噬中的作用。體內(nèi)實驗中,通過高脂喂養(yǎng)ApoE-/-小鼠20周構(gòu)建動脈粥樣硬化晚期斑塊模型。然后隨機(jī)分為兩個組,分別通過灌胃給予生理鹽水和人參皂苷Rb1(50 mg/kg/d),隔天一次給藥。干預(yù)7周后,病理學(xué)方法檢測斑塊脂質(zhì)沉積、巨噬細(xì)胞浸潤、平滑肌數(shù)量以及膠原含量,并通過分子學(xué)檢測斑塊中自噬蛋白LC3B和SQSTM1表達(dá)及AMPK磷酸化,探究Rb1影響斑塊穩(wěn)定性的可能機(jī)制。結(jié)果1.Rb1在20-80 μ范圍內(nèi)濃度依賴性地降低了泡沫細(xì)胞內(nèi)的脂質(zhì)沉積。2.Rb1處理后細(xì)胞LC3Ⅱ/LC3Ⅰ比例增加、SQSTM1降低,并且細(xì)胞中自噬雙層膜結(jié)構(gòu)增多,提示Rb1促進(jìn)了細(xì)胞的自噬水平。3.經(jīng)慢病毒轉(zhuǎn)染Atg5 siRNA抑制自噬后,Rb1降低泡沫細(xì)胞脂質(zhì)沉積的作用明顯減弱。4.Rb1促進(jìn)了 AMPK磷酸化,加入AMPK抑制劑compound C后,Rb1激活自噬的作用減弱。5.與對照組相比,Rb1處理組小鼠斑塊自噬蛋白LC3Ⅱ/LC3Ⅰ比例增加、SQSTM1降低,并且AMPK磷酸化增加。6.與對照組相比,Rb1處理組小鼠斑塊脂質(zhì)沉積和巨噬細(xì)胞浸潤減少,膠原和平滑肌數(shù)量增多,斑塊穩(wěn)定性增加。結(jié)論Rb1通過激活巨噬細(xì)胞自噬促進(jìn)了動脈粥樣硬化斑塊脂質(zhì)代謝和斑塊穩(wěn)定;AMPK通路在Rb1激活自噬中發(fā)揮重要作用。
[Abstract]:Background Atherosclerotic plaque rupture and thrombosis are the main pathological mechanisms leading to acute cardiovascular and cerebrovascular events. Vulnerable plaques for potentially ruptured plaques. Early and active intervention is an important method to prevent and reduce the incidence of acute cardiovascular and cerebrovascular events. As is a chronic inflammatory disease driven by lipid. Its main pathological manifestations are lipid accumulation and inflammatory cell infiltration. Macrophages play an important role in this process. Low density lipoprotein cholesterol in the blood is swallowed by macrophages and is found in the form of cholesterol ester in lipid droplets. Macrophages that accumulate large amounts of lipid become foam cells. How to reduce lipid accumulation in foam cells, for reducing plaque lipid load. It is important to promote plaque stability. Ginsenoside Rb1 is the most active ingredient in ginseng. Current studies have found that Rb1 can reduce blood lipids, reduce body weight and resist inflammation. Inhibition of angiogenesis and enhancement of insulin sensitivity and other physiological effects. However, the role of Rb1 in atherosclerosis has not been studied. This study intends to construct as mouse model and culture macrophages in vitro. To investigate the effect of ginsenoside Rb1 on the stability of atherosclerotic plaque by macrophage autophagy and its related mechanism. Objective to establish a foam cell model in vitro and a vulnerable plaque model in atherosclerotic mice. . To study the molecular mechanism of ginsenoside Rb1 regulating macrophage autophagy, affecting lipid metabolism and stabilizing as vulnerable plaque. Methods in vitro, we extracted primary peritoneal macrophages from C57BL / 6 mice. The foam cells were induced by ox-LDL(100 渭 g / ml for 24 hours, then treated with different concentrations of Rb1 for 24 hours. Oil red O staining was used to detect lipid deposition in each group. The contents of autophagy protein LC3 鈪，

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