氧化应激环境下miR-155通过自噬调控内皮细胞功能的研究

发布时间：2018-07-14 12:48

【摘要】：研究背景与研究目的:目前冠心病、高血压等慢性血管性疾病发病率逐年增加,防治工作极为重要。内皮细胞功能紊乱是血管性疾病发生、发展的重要病理基础。血管内脂质代谢失调及缺氧等多种原因导致内皮细胞的损伤,血液与血管之间的屏障作用减弱,造成血小板黏附聚集,血管痉挛,血栓形成,血管腔狭窄。因此,保护及维持血管内皮细胞的正常功能,促进受损内皮细胞的增殖及提高细胞存活率是目前慢性损伤性血管疾病的研究热点。微小RNA(mircoRNA)是一种典型的多功能非编码RNA,能在转录水平降解或者抑制靶标mRNA的表达,调控细胞的生物功能,与细胞生成,炎症及肿瘤发展等多种病理生理相关。自噬是细胞内一种保护性程序,能够消化降解细胞内损伤的细胞器来维持细胞内环境稳态。微小RNA通过自噬对动脉粥样硬化的发生与进展的研究已有报道,而其中miR-155在损伤内皮细胞的作用研究甚少。本课题拟讨论miR-155对氧化应激条件下受损内皮细胞功能的影响,并探讨其可能调控机制。方法:1、在SicenceCELL细胞库中购买人脐静脉内皮细胞株(HUVECs),双氧水(H2O2)处理细胞后构建氧化应激细胞模型,通过检测细胞内毒性物质氧自由基的生成量及自噬程度等指标对细胞模型进行鉴定;2、在成功复制体外氧化应激细胞模型上,利用质粒转染miR-155,监测转染后细胞毒性物质的生成量及细胞增殖存活率的影响。同时采用RT-PCR和Western blotting、共聚焦免疫荧光技术检测损伤后细胞内的自噬发生的程度;3、探索ATG5在miR-155调控功能紊乱的内皮细胞的作用。结果:1、通过对不同浓度时间的H2O2摸索,在400nmol/l浓度12小时处理的内皮细胞氧自由基生成量趋于平稳,而自噬水平最高。利用此浓度及时间点复制体外氧化应激细胞模型。2、抑制miR-155表达后损伤的内皮细胞内自噬程度增高,细胞内活性氧ROS生成减少,细胞存活率增加。3、miR-155通过调控ATG5的表达来调节内皮细胞自噬水平来影响内皮细胞的功能。结论:miR-155通过自噬减少细胞毒性物质ROS的生成,提高氧化应激条件下受损内皮细胞的存活率;其中,自噬的调控作用可能是通过ATG5通路来实现。
[Abstract]:Background and objective: the incidence of coronary heart disease, hypertension and other chronic vascular diseases is increasing year by year. Endothelial dysfunction is an important pathological basis for the occurrence and development of vascular diseases. The dysfunction of lipid metabolism in blood vessel and anoxia lead to the injury of endothelial cells and the weakening of the barrier between blood and blood vessel, resulting in platelet adhesion, vasospasm, thrombosis and stenosis of blood vessel. Therefore, protecting and maintaining the normal function of vascular endothelial cells, promoting the proliferation of injured endothelial cells and increasing the cell survival rate are the research focus of chronic vascular diseases. MicroRNA (mircoRNA) is a typical multifunctional non-coding RNAs that can degrade or inhibit the expression of target mRNA at the transcriptional level, regulate the biological function of cells, and be related to many pathophysiology, such as cell formation, inflammation and tumor development. Autophagy is a protective procedure in cells that can digest organelles that degrade intracellular damage to maintain the homeostasis of the intracellular environment. MicroRNA has been reported on the pathogenesis and progression of atherosclerosis through autophagy, but the role of miR-155 in the injury of endothelial cells is rarely studied. The purpose of this study was to investigate the effects of miR-155 on the function of injured endothelial cells under oxidative stress and its possible regulatory mechanisms. Methods Cell models of oxidative stress were established by treating human umbilical vein endothelial cell line (HUVECs) with hydrogen peroxide (H _ 2O _ 2) in the Sicence cell bank. The cell model was identified by detecting the amount of oxygen free radicals and the degree of autophagy. The effects of transfection of miR-155 on the production of cytotoxic substances and the survival rate of cell proliferation were monitored. At the same time, RT-PCR and Western blotting were used to detect the degree of autophagy after injury by confocal immunofluorescence, and to explore the role of ATG5 in endothelial cells with miR-155 regulatory dysfunction. Results the oxygen free radical production of endothelial cells treated with 400nmol/l for 12 hours tended to be stable and autophagy was the highest by groping for H2O2 at different concentrations. In vitro oxidative stress cell model was established at this concentration and time point. After inhibiting the expression of miR-155, the degree of intracellular autophagy was increased, and the production of reactive oxygen species (Ros) was decreased. Cell survival increased. 3miR-155 affected endothelial cell function by regulating the expression of ATG5 to regulate the level of endothelial autophagy. Conclusion the fraction miR-155 reduces the production of Ros by autophagy and increases the survival rate of injured endothelial cells under oxidative stress, and the regulation of autophagy may be mediated by ATG5 pathway.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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