邻苯二甲酸（2-乙基已基）酯对原代培养的大鼠海马神经元的毒性研究

发布时间：2018-05-18 01:13

本文选题：大鼠 + 海马神经元　；参考：《山东师范大学》2012年硕士论文

【摘要】：2011年5月台湾地区发生“塑化剂”事件，这一事件的元凶——邻苯二甲酸（2—乙基己基）酯(DEHP)得到了全社会空前的关注。DEHP在工业上有广泛的应用，常用来增强塑料的可塑性，主要用在聚氯乙烯（PVC）塑料制品中，在保鲜膜、食品包装材料、玩具以及医疗器械用品中均有应用。在这次事件中DEHP被不法商家用在食品饮料中以代替食品添加剂“起云剂”，这是近年来台湾最严重的食品安全事件。此外，对于添加了DEHP的PVC塑料制品做成的食品包装材料，DEHP也可能从食品包装材料中迁移到食品或水中。人们对于DEHP对动物和人体的毒性已经有广泛的研究，除了DEHP对生殖和发育系统的影响以外，也有大量实验结果表明DEHP可通过氧化损伤对不同种系的体外培养的细胞产生毒性作用。细胞的氧化损伤是氧化和抗氧化作用失衡的结果，氧化损伤是引起组织和细胞凋亡的一个重要机制。人体脑组织中含有的与氧化有关的物质水平较高，同时脑内抗氧化酶及非酶的自由基清除剂含量很低，使得脑组织极容易受到自由基损伤，脑内神经元的死亡与多种神经退行性疾病有关。大脑海马是学习和记忆的主要场所，海马神经元的损伤与阿尔茨海默病（Alzheimer’s disease,AD）有密切关系。本论文重点研究DEHP对体外培养的海马神经元的毒性作用，探讨其对中枢神经系统的影响。实验目的：从细胞水平上研究DEHP对海马神经元的毒性作用，从而揭示DEHP对中枢神经系统可能的影响，为DEHP的安全性评价提供一定的数据支持。实验方法：采用无血清培养方法进行Wistar大鼠胎鼠海马神经元的培养，在倒置荧光显微镜下观察海马神经元生长状态，及DEHP对神经元形态的影响。根据相关的文献中DEHP对细胞起到毒性作用的浓度摸索DEHP引起海马神经元毒性的浓度，用MTT法研究DEHP对海马神经元活力的影响。通过荧光染料Hoechst 33258染色观察，及凋亡酶Caspase-3活力测定，研究海马神经元的凋亡。用荧光染料DCFH-DA对培养海马神经元进行荧光染色，通过激光扫描共聚焦显微镜分析细胞内ROS的水平，并测定神经元超氧化物歧化酶（SOD）活力及谷胱甘肽（GSH）的含量，探究DEHP对海马神经元的氧化损伤。实验结果：与用胎牛血清培养的神经元相比，无血清培养能得到纯度较高并且生长状态良好的神经元。经过浓度摸索，，最终确定试验中采用的DEHP浓度分别为0.25mM，0.5mM，1mM，2mM，3mM。形态观察和MTT实验结果表明DEHP造成海马神经元形态的改变和细胞活力的下降，并且这一影响与DEHP浓度具有量-效关系，其中3mM作用组细胞活力为对照组的52%。Hoechst 33258染色后观察发现，细胞DNA皱缩，并出现凋亡小体，且Caspase-3活性显著升高，表明DEHP诱导海马神经元出现凋亡。DEHP导致神经元产生大量ROS，同时有SOD活力下降及GSH表达量下降，对海马神经元造成氧化损伤。
[Abstract]:In May 2011, the "plasticizer" incident occurred in Taiwan. The main culprit of the incident, DEHPphthalate (2-ethylhexyl) phthalate), has received unprecedented attention from the whole society. DEHP has been widely used in industry and is often used to enhance the plasticity of plastics. Mainly used in PVC PVC plastic products, fresh film, food packaging materials, toys and medical devices are used. In the incident, DEHP was used by illegal merchants to replace the food additive "cloud lifting agent" in food and beverage, which is the most serious food safety incident in Taiwan in recent years. In addition, DEHP may be transferred from food packaging materials to food or water for PVC plastic products with DEHP added. The toxicity of DEHP to animals and humans has been extensively studied, in addition to the effects of DEHP on the reproductive and developmental systems. A large number of experimental results also showed that DEHP can produce toxic effects on the cultured cells of different strains through oxidative damage. Oxidative damage of cells is the result of imbalance of oxidation and antioxidation, and oxidative damage is an important mechanism to induce apoptosis of tissues and cells. There are high levels of oxidation-related substances in human brain tissues, and low levels of antioxidant enzymes and non-enzymatic free radical scavengers in the brain, making brain tissue extremely vulnerable to free radical damage. The death of neurons in the brain is associated with a variety of neurodegenerative diseases. Hippocampus is the main site of learning and memory. The damage of hippocampal neurons is closely related to Alzheimer's disease (AD). This paper focuses on the toxic effects of DEHP on cultured hippocampal neurons and its effects on the central nervous system (CNS). Objective: to study the toxicity of DEHP to hippocampal neurons at the cellular level, so as to reveal the possible effects of DEHP on the central nervous system and to provide some data support for the safety evaluation of DEHP. Methods: the hippocampal neurons of fetal Wistar rats were cultured by serum-free culture. The growth of hippocampal neurons and the effect of DEHP on the morphology of neurons were observed under inverted fluorescence microscope. According to the concentration of cytotoxic effect of DEHP on hippocampal neurons induced by DEHP, the effect of DEHP on the activity of hippocampal neurons was studied by MTT method. The apoptosis of hippocampal neurons was studied by fluorescence dye Hoechst 33258 staining and the activity of apoptotic enzyme Caspase-3. The cultured hippocampal neurons were stained with fluorescent dye DCFH-DA. The level of ROS was analyzed by laser scanning confocal microscopy, and the activity of superoxide dismutase (SOD) and the content of glutathione (GSH) were measured. To investigate the oxidative damage of DEHP to hippocampal neurons. Results: compared with those cultured with fetal bovine serum, serum-free culture could obtain high purity and good growth state of neurons. The concentration of DEHP in the experiment was determined to be 0.25mMU 0.5mMU 1 mMU 2mMU 3mM respectively. Morphological observation and MTT experiment showed that DEHP caused morphological changes and decreased cell viability of hippocampal neurons, and this effect had a dose-effect relationship with the concentration of DEHP. The cell viability in the 3mM group was observed after 52%.Hoechst 33258 staining in the control group. DNA shrinked and apoptotic corpuscles appeared, and the activity of Caspase-3 increased significantly, which indicated that DEHP induced apoptosis of hippocampal neurons. DEHP led to a large number of ROSs in hippocampal neurons. At the same time, the activity of SOD and the expression of GSH decreased, which caused oxidative damage to hippocampal neurons.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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