塑化剂有毒代谢物MEHP诱导自噬参与胰岛INS-1细胞氧化性DNA损伤机制探讨

发布时间：2018-01-18 15:22

本文关键词：塑化剂有毒代谢物MEHP诱导自噬参与胰岛INS-1细胞氧化性DNA损伤机制探讨　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:邻苯二甲酸单(2-乙基己基)酯(Mono-(2-ethylhexyl)phthalate,MEHP)是邻苯二甲酸(2-乙基己基)酯(di-(2-ethylhexyl)phthalate,DEHP)的有效毒性代谢产物。由于塑料产品中和日常饮食中DEHP越来越多的被使用,所以DEHP和其水解物MEHP对人体的伤害值得重视。已经有研究表明邻苯二甲酸酯类代谢物与很多慢性疾病有关,像肥胖和二型糖尿病等。而累积DNA损伤和一些氧化性损伤与胰岛素抵抗和肥胖的发生都有直接关系。近年来发现在DNA损伤的发生过程中还会检测到自噬的发生,自噬的出现对细胞是有利还是有害不是完全清楚,其间作用的信号通路也不是很明朗。白藜芦醇作为抗氧化剂可以消除细胞内活性氧,达到化学预防的作用,对肥胖和糖尿病等慢性病有着积极的治疗作用。所以在本实验中,探究MEHP对INS-1细胞造成的DNA损伤,分析造成DNA损伤的机制途径,同时还探究自噬的发生与DNA损伤的关系及其对细胞的影响;选用白藜芦醇作为保护剂,观察其在MEHP产生细胞毒性过程中的作用。方法:INS-1细胞被选为研究对象。MTT实验用来检测MEHP对INS-1细胞的毒性作用和对细胞生存率的影响;彗星实验用来评定MEHP对INS-1细胞的遗传毒性、DNA损伤水平;实时荧光定量PCR检测INS-1细胞内DNA损伤相关基因ATM和P53的表达量变化。检测细胞内ROS变化使用2',7'-二氯二氢荧光素荧光染料(DCFH-DA),检测溶酶体膜稳定性用吖啶橙(AO),检测线粒体膜电位变化用JC-1染色,使用分光光度计和荧光显微镜观察拍照。用蛋白印迹法检测相关蛋白表达量变化。检测白藜芦醇和自噬抑制剂的作用都需要预先对细胞进行处理,再加MEHP作用。结果:与对照组相比,MEHP作用后INS-1细胞产生明显的DNA损伤,表现为彗星实验中细胞拖尾现象加剧等,MEHP还导致细胞内活性氧(ROS)水平升高、溶酶体膜稳定性下降、溶酶体内组织蛋白酶B释放增多、线粒体膜电位崩溃以及自噬蛋白表达增加。经白藜芦醇预处理后上述现象均减轻,经自噬抑制剂预处理后自噬蛋白表达减少,细胞内ROS水平反而增多。结论:MEHP对INS-1细胞具有遗传毒性。MEHP诱导产生的INS-1细胞DNA损伤是氧化应激造成的。MEHP诱导产生的INS-1细胞DNA损伤跟溶酶体-线粒体轴损伤有关。MEHP诱导产生INS-1细胞DNA损伤同时也诱导细胞自噬增加,而自噬对INS-1细胞在DNA损伤过程中起到一种保护作用。白藜芦醇可以通过减轻氧化应激和恢复溶酶体-线粒体轴的稳定性来减少MEHP对INS-1细胞造成的DNA损伤。
[Abstract]:Objective: to investigate the effect of mono-2-ethylhexylphthalate on mono-2-ethylhexylphthalate of phthalate. MEHP is a kind of di- (2-ethylhexylphthalate) phthalate (2-ethylhexylphthalate) phthalate of phthalate (2-ethylhexylphthalate). Due to the increasing use of DEHP in plastic products and diet. Therefore, the harm of DEHP and its hydrolysate MEHP to human body is worthy of attention. Some studies have shown that phthalate metabolites are related to many chronic diseases. Such as obesity and type 2 diabetes. Cumulative DNA damage and some oxidative damage are both directly related to insulin resistance and obesity. Autophagy has also been found in the process of DNA damage in recent years. What happened. Whether autophagy is beneficial or harmful to cells is not completely clear, and the signaling pathway is not clear. Resveratrol as an antioxidant can eliminate reactive oxygen species in cells to achieve chemoprevention. It has a positive effect on chronic diseases such as obesity and diabetes. So in this experiment, we explore the DNA damage caused by MEHP on INS-1 cells and analyze the mechanism of DNA damage. At the same time, the relationship between autophagy and DNA damage and its effect on cells were explored. Resveratrol was used as protective agent. To observe the role of MEHP in the process of cytotoxicity. Methods:. INS-1 cells were selected as the object of study. MTT assay was used to detect the toxicity of MEHP to INS-1 cells and the effect of MEHP on cell survival. Comet assay was used to evaluate the damage level of MEHP to INS-1 cells. The expression of DNA damage related genes ATM and p53 in INS-1 cells was detected by real-time fluorescence quantitative PCR. The changes of ROS in INS-1 cells were detected by 2'. The stability of lysosomal membrane was detected by acridine orange (Acridine orange), and the change of mitochondrial membrane potential was stained with JC-1. Spectrophotometer and fluorescence microscope were used to observe and photograph. Western blotting was used to detect the expression of related proteins. The effects of resveratrol and autophagy inhibitor were all pre-treated. Results: compared with the control group, the DNA damage of INS-1 cells was obvious, which showed that the tail phenomenon was aggravated in comet assay. MEHP also increased the level of intracellular reactive oxygen species (Ros), decreased the stability of lysosomal membrane and increased the release of cathepsin B in lysosome. The mitochondrial membrane potential collapse and autophagy protein expression increased. After resveratrol pretreatment, the above phenomenon was alleviated, and the autophagy protein expression decreased after autophagy inhibitor pretreatment. The level of intracellular ROS increased. Conclusion:. Genotoxicity of MEHP to INS-1 cells. MEHP-induced DNA damage of INS-1 cells is induced by oxidative stress. MEHP-induced DNA damage of INS-1 cells. MEHP-induced DNA damage in INS-1 cells and increased autophagy. Resveratrol can reduce the effect of MEHP on INS- by reducing oxidative stress and restoring the stability of lysosomal mitochondrial axis. 1 DNA damage caused by cells.
【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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