邻苯二甲酸—单—乙基己酯对EA.hy926细胞的毒性作用及分子机制研究

发布时间：2018-06-10 07:06

本文选题：邻苯二甲酸-单-乙基己酯 + 自噬　；参考：《大连医科大学》2017年硕士论文

【摘要】：目的:邻苯二甲酸-单-乙基己酯(Mono-(2-ethylhexyl)phthalate,MEHP)是邻苯二甲酸二乙基己酯(Di-2-ethylhexyl phthalate,DEHP)的活性代谢产物。DEHP是邻苯二甲酸酯类化合物(Phthalate esters,PAEs)中应用最为普遍的一种人工合成的有机化合物,可以显著提高塑料制品的可塑性和柔韧性,被广泛地应用到医疗用品当中。DEHP进入人体后很快经肝脏和肾脏代谢成MEHP。早期的研究大部分关注MEHP对肝脏、肾脏、生殖系统和生长发育毒性,MEHP还具有心血管系统毒性,但是其分子机制尚未完全阐明。本实验以EA.hy926细胞为研究对象,探讨MEHP作用于EA.hy926细胞后能否通过活性氧自由基(Reactive oxygen species,ROS)诱导自噬。方法:研究MEHP作用于EA.hy926细胞后是否通过ROS诱导自噬的发生,应用MTT法(四甲基偶氮唑盐微量酶反应比色法)检测不同浓度(0μM-1600μM)MEHP处理后的EA.hy926细胞存活率的变化;使用不同试剂自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3-MA)、自噬激活剂雷帕霉素、活性氧抑制剂N-乙酰半胱氨酸(NAC)、si RNA Akt1、Akt1激活剂胰岛素预处理后,用不同浓度(0μM-400μM)MEHP处理EA.hy926细胞,检测细胞存活率的变化;用透射电子显微镜观察细胞自噬小体的超微结构;用Western blot法(蛋白质免疫印迹法)检测不同浓度(0μM-200μM)MEHP处理及NAC预处理后P62和LC3(细胞内自噬标记物-微管相关蛋白轻链3)蛋白表达水平的变化;用DCFH-DA(2’,7’-二氢二氯荧光素)检测不同浓度(0μM-200μM)MEHP处理及NAC预处理后细胞内ROS的含量变化;用荧光显微镜观察经JC-1染色后EA.hy926细胞内线粒体膜电位的改变情况;用免疫荧光方法检测不同浓度(0μM-200μM)MEHP处理后细胞内p-Akt1水平的变化;使用不同试剂(NAC、si RNA Akt1、胰岛素)预处理后,检测细胞内p-Akt1水平的变化;用Western blot法检测si RNA Akt1、胰岛素预处理后的LC3蛋白表达水平的变化。结果:经过不同浓度MEHP(0μM-400μM)染毒24 h后,随着MEHP浓度的增加EA.hy926细胞存活率显著下降;用3-MA、NAC和胰岛素干预后,细胞存活率明显增高;用雷帕霉素、si RNA Akt1干预后,细胞存活率明显下降。在透射电子显微镜下可观察到自噬小体,且数量增加;LC3-II和P62蛋白表达随MEHP浓度增加而增加,这说明聚集的自噬小体是由于自噬小体降解障碍引起的,用NAC预处理后,LC3-II蛋白表达量减少;细胞内ROS水平随MEHP剂量的升高而升高,用NAC预处理后可有效降低ROS水平;线粒体膜电位随MEHP浓度的升高而下降,用NAC预处理后可显著升高使线粒体膜电位;MEHP处理后细胞内p-Akt1水平下降,用NAC、胰岛素预处理后p-akt1水平升高,用si RNA Akt1干预后,p-Akt1水平显著下降;用胰岛素预处理后,LC3-II蛋白表达水平降低,而si RNA Akt1干预后则增高。结论:MEHP作用于EA.hy926细胞后,可促使细胞内自噬小体的形成,MEHP主要依赖于ROS通过Akt1通路诱导自噬性细胞死亡。
[Abstract]:Objective: the active metabolite of diethylhexyl phthalate (Di-2-ethylhexatehp) is the most common synthetic organic compound of phthalate ester, Phthalate estersPAeses, which is one of the most common metabolites of diethylhexyl phthalate (Di-2-ethylhexatehexateDEHPP), which is the most common synthetic organic compound of phthalic acid-monoethylhexylhexylphthalate (MEHP), which is a kind of diethylhexyl phthalate (diethylhexyl) phthalate (DEHP). It can significantly improve the plasticity and flexibility of plastic products and is widely used in medical supplies. DEHP is quickly metabolized into MEHPthrough liver and kidney after entering into human body. Most early studies have focused on the cardiovascular toxicity of MEHP to liver, kidney, reproductive system and growth and development, but its molecular mechanism has not been fully elucidated. In this study, EA.hy926 cells were studied to investigate whether MEHP could induce autophagy by reactive oxygen species-ROS after it was treated with EA.hy926 cells. Methods: to study whether MEHP could induce autophagy in EA.hy926 cells by Ros. MTT assay was used to detect the survival rate of EA.hy926 cells treated with different concentrations of MEHP (0 渭 M-1600 渭 MMEHP). EA.hy926 cells were treated with different reagent autophagy inhibitor 3-Methyladenine 3-MAHp, autophagy activator rapamycin, active oxygen inhibitor N-acetylcysteine (Nacetylcysteine) -nacylcysteine siRNA Akt1 / Akt1 activator insulin, and EA.hy926 cells were treated with different concentrations of 0 渭 M-400 渭 MMEHP. The ultrastructure of autophagy was observed by transmission electron microscope. The expression levels of P62 and LC3 (microtubule-associated protein light chain 3) were detected by Western blot (Western blot) after different concentrations of 0 渭 M-200 渭 MMEHP and NAC pretreatment. The changes of Ros content in EA.hy926 cells treated with different concentrations of 0 渭 M-200 渭 MMEHP and pretreatment with NAC were detected by DCFH-DAH2-Dichlorofluorescein, and the changes of mitochondrial membrane potential in EA.hy926 cells were observed by fluorescence microscope. The changes of p-Akt1 levels in cells treated with different concentrations of 0 渭 M-200 渭 MMEHP were detected by immunofluorescence method, and the changes of p-Akt1 levels in cells were detected after pretreatment with different reagent NACSI RNA Akt1 (insulin). The expression of si RNA Akt1 and LC3 protein after insulin preconditioning was detected by Western blot assay. Results: the survival rate of EA.hy926 cells decreased significantly with the increase of MEHP concentration 24 h after exposure to different concentrations of MEHP0 渭 M-400 渭 M, the survival rate of EA.hy926 cells increased significantly after treated with 3-MAG NAC and insulin, and the survival rate of EA.hy926 cells significantly decreased after treated with rapamycin siRNA Akt1. Autophagy bodies were observed under transmission electron microscope, and the expression of LC3-II and P62 proteins increased with the increase of MEHP concentration, which suggested that the aggregation of autophagy bodies was caused by the degradation of autophagy bodies. After pretreatment with NAC, the expression of LC3-II protein decreased, the intracellular Ros level increased with the increase of MEHP dose, the Ros level decreased effectively after NAC pretreatment, and the mitochondrial membrane potential decreased with the increase of MEHP concentration. Pretreatment with NAC significantly increased the level of p-Akt1, increased the level of p-akt1 after pretreatment with NAC and insulin, and decreased the level of p-Akt1 after treatment with siRNA Akt1. The expression of LC3-II protein decreased after insulin preconditioning, but increased after the intervention of siRNA Akt1. Conclusion the formation of autophagy bodies in EA.hy926 cells induced by the action of 1: MEHP is mainly dependent on Ros inducing autophagic cell death through Akt1 pathway.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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