Ox-LDL诱导早期巨噬细胞源泡沫细胞形成和损伤中相关动力学机制研究

发布时间：2018-05-01 17:32

本文选题：Ox-LDL + 诱导　；参考：《浙江大学》2006年博士论文

【摘要】：动脉粥样硬化(Atherosclerosis,AS)是一种多因素疾病,与多种危险因子包括血脂紊乱、糖尿病、肥胖和高血压有关,重要的基本病理改变是氧化低密度脂蛋白(oxidized low-density lipoprotein,Ox-LDL)在血管壁的积聚,其中巨噬细胞在血管壁的脂质代谢中起着关键而复杂的作用。在早期脂质浸润中,巨噬细胞通过清道夫受体(CD36等)摄取Ox-LDL,形成泡沫细胞,大量积聚的Ox-LDL引起细胞毒性使细胞产生大量炎症因子,改变了细胞的形态和信号转导,进而损伤细胞影响动脉粥样硬化的发展。因此,早期AS最基本的病理特征之一就是巨噬源泡沫细胞的形成和损伤,但目前其形成和损伤的时间和空间动力学机理仍不清楚。本研究以PMA诱导人单核细胞U937源的巨噬细胞为靶细胞,通过Ox-LDL诱导的巨噬细胞源泡沫细胞模型,利用流式细胞术、激光共聚焦显微术和分子生物学技术等实验技术,结合动力学分析方法,研究了动脉粥样硬化早期巨噬细胞源泡沫细胞形成和损伤的相关动力学机制。本研究分别在Ox-LDL急性和慢性刺激等不同条件下,对影响泡沫细胞形成的部分关键因子胆固醇酯(CE)、总胆固醇(TC)、CD36、CD54和F-actin,以及[Ca~(2+)]i等进行了时间和空间上的相关动力学研究。研究结果显示,1)在慢性刺激下,Ox-LDL能显著以时间依赖的方式诱导细胞内CE和TC的升高(P0.01),尤其在24h时(CE/TC)%50%,表明了Ox-LDL诱导早期泡沫细胞形成的时间动力学变化,即Ox-LDL刺激24h后形成典型巨噬细胞源泡沫细胞;2)在急性和慢性刺激下,Ox-LDL能显著诱导[Ca~(2+)]i的升高,但在急性刺激时,胞外液无论在有无Ca~(2+)存在的情况下,[Ca~(2+)]i都呈不同程度的升高,且在空间上形成明显的跨膜Ca~(2+)梯度变化;3)在慢性刺激下,Ox-LDL以时间依赖的方式诱导细胞表面CD54和CD36表达的升高,以及F-actin时间和空间上多聚化的改变; 另外,本研究还分别在Ox-LDL急性和慢性刺激等不同条件下,对影响泡沫细胞内线粒体损伤的部分关键因子H_2O_2、NO和O_2~(·-)、线粒体呼吸、线粒体膜电位(△ψm)、谷胱甘肽(GSH)和Bcl-2蛋白的表达、以及细胞的凋亡和坏死等进行了相关动力学研究。研究结果显示,1)在急性和慢性刺激下,Ox-LDL能显著
[Abstract]:Atherosclerotic atherosclerosis is a multifactorial disease associated with multiple risk factors including dyslipidemia, diabetes, obesity and hypertension. An important underlying pathological change is the accumulation of oxidized low density lipoprotein (low-density) protein Ox-LDLs in the vascular wall. Macrophages play a key and complex role in lipid metabolism in vascular walls. In the early stage of lipid infiltration, macrophages ingest Ox-LDLthrough scavenger receptor CD36 and form foam cells. The accumulation of Ox-LDL leads to the production of a large number of inflammatory factors and changes the morphology and signal transduction of the cells. And then damage the cells to affect the development of atherosclerosis. Therefore, one of the most basic pathological features of early as is the formation and injury of macrophage foam cells, but the temporal and spatial dynamic mechanism of the formation and injury is still unclear. In this study, macrophages derived from human monocyte U937 induced by PMA were used as target cells, and Ox-LDL induced macrophage foam cell model was used. Flow cytometry, laser confocal microscopy and molecular biology techniques were used. The kinetic mechanism of macrophage-derived foam cell formation and injury in early atherosclerosis was studied by means of kinetic analysis. In this study, under different conditions of acute and chronic stimulation of Ox-LDL, some key factors affecting foam cell formation, such as cholesterol ester, total cholesterol (TCC) CD36, CD54 and F-actini, and [Ca~(2] I, were studied in time and space respectively. The results showed that Ox-LDL could significantly induce the increase of CE and TC in cells in a time-dependent manner, especially at 24 h, which indicated that Ox-LDL induced early foam cell formation in a time-dependent manner. That is to say, after 24 hours of Ox-LDL stimulation, typical macrophage derived foam cells were formed. Ox-LDL significantly induced the increase of [Ca~(2] I under acute and chronic stimulation, but in acute stimulation, [Ca~(2] I increased in varying degrees, regardless of the presence of Ca~(2 in the extracellular fluid. Furthermore, a significant transmembrane Ca~(2 gradient change was formed in space. Under chronic stimulation, Ox-LDL induced the increase of CD54 and CD36 expression on the surface of cells in a time-dependent manner, as well as the changes of F-actin in time and space. In addition, under different conditions such as acute and chronic Ox-LDL stimulation, the expression of H _ 2O _ 2NO and O _ 2C, mitochondrial respiration, mitochondrial membrane potential, glutathione (GSH) and Bcl-2 protein in foam cells were also studied. The kinetics of apoptosis and necrosis were studied. The results showed that 1) Ox-LDL was significant in acute and chronic stimuli.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363

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