本文選題：大鼠 + 海馬神經(jīng)元��； 參考：《山東師范大學(xué)》2012年碩士論文

【摘要】：2011年5月臺(tái)灣地區(qū)發(fā)生“塑化劑”事件，這一事件的元兇——鄰苯二甲酸（2—乙基己基）酯(DEHP)得到了全社會(huì)空前的關(guān)注。DEHP在工業(yè)上有廣泛的應(yīng)用，常用來增強(qiáng)塑料的可塑性，主要用在聚氯乙烯（PVC）塑料制品中，在保鮮膜、食品包裝材料、玩具以及醫(yī)療器械用品中均有應(yīng)用。在這次事件中DEHP被不法商家用在食品飲料中以代替食品添加劑“起云劑”，這是近年來臺(tái)灣最嚴(yán)重的食品安全事件。此外，對(duì)于添加了DEHP的PVC塑料制品做成的食品包裝材料，DEHP也可能從食品包裝材料中遷移到食品或水中。 人們對(duì)于DEHP對(duì)動(dòng)物和人體的毒性已經(jīng)有廣泛的研究，除了DEHP對(duì)生殖和發(fā)育系統(tǒng)的影響以外，也有大量實(shí)驗(yàn)結(jié)果表明DEHP可通過氧化損傷對(duì)不同種系的體外培養(yǎng)的細(xì)胞產(chǎn)生毒性作用。細(xì)胞的氧化損傷是氧化和抗氧化作用失衡的結(jié)果，氧化損傷是引起組織和細(xì)胞凋亡的一個(gè)重要機(jī)制。 人體腦組織中含有的與氧化有關(guān)的物質(zhì)水平較高，同時(shí)腦內(nèi)抗氧化酶及非酶的自由基清除劑含量很低，使得腦組織極容易受到自由基損傷，腦內(nèi)神經(jīng)元的死亡與多種神經(jīng)退行性疾病有關(guān)。大腦海馬是學(xué)習(xí)和記憶的主要場(chǎng)所，海馬神經(jīng)元的損傷與阿爾茨海默病（Alzheimer’s disease,AD）有密切關(guān)系。本論文重點(diǎn)研究DEHP對(duì)體外培養(yǎng)的海馬神經(jīng)元的毒性作用，探討其對(duì)中樞神經(jīng)系統(tǒng)的影響。 實(shí)驗(yàn)?zāi)康模簭募?xì)胞水平上研究DEHP對(duì)海馬神經(jīng)元的毒性作用，從而揭示DEHP對(duì)中樞神經(jīng)系統(tǒng)可能的影響，為DEHP的安全性評(píng)價(jià)提供一定的數(shù)據(jù)支持。 實(shí)驗(yàn)方法：采用無血清培養(yǎng)方法進(jìn)行Wistar大鼠胎鼠海馬神經(jīng)元的培養(yǎng)，在倒置熒光顯微鏡下觀察海馬神經(jīng)元生長狀態(tài)，及DEHP對(duì)神經(jīng)元形態(tài)的影響。根據(jù)相關(guān)的文獻(xiàn)中DEHP對(duì)細(xì)胞起到毒性作用的濃度摸索DEHP引起海馬神經(jīng)元毒性的濃度，用MTT法研究DEHP對(duì)海馬神經(jīng)元活力的影響。通過熒光染料Hoechst 33258染色觀察，及凋亡酶Caspase-3活力測(cè)定，研究海馬神經(jīng)元的凋亡。用熒光染料DCFH-DA對(duì)培養(yǎng)海馬神經(jīng)元進(jìn)行熒光染色，通過激光掃描共聚焦顯微鏡分析細(xì)胞內(nèi)ROS的水平，并測(cè)定神經(jīng)元超氧化物歧化酶（SOD）活力及谷胱甘肽（GSH）的含量，探究DEHP對(duì)海馬神經(jīng)元的氧化損傷。 實(shí)驗(yàn)結(jié)果：與用胎牛血清培養(yǎng)的神經(jīng)元相比，無血清培養(yǎng)能得到純度較高并且生長狀態(tài)良好的神經(jīng)元。經(jīng)過濃度摸索，，最終確定試驗(yàn)中采用的DEHP濃度分別為0.25mM，0.5mM，1mM，2mM，3mM。形態(tài)觀察和MTT實(shí)驗(yàn)結(jié)果表明DEHP造成海馬神經(jīng)元形態(tài)的改變和細(xì)胞活力的下降，并且這一影響與DEHP濃度具有量-效關(guān)系，其中3mM作用組細(xì)胞活力為對(duì)照組的52%。Hoechst 33258染色后觀察發(fā)現(xiàn)，細(xì)胞DNA皺縮，并出現(xiàn)凋亡小體，且Caspase-3活性顯著升高，表明DEHP誘導(dǎo)海馬神經(jīng)元出現(xiàn)凋亡。DEHP導(dǎo)致神經(jīng)元產(chǎn)生大量ROS，同時(shí)有SOD活力下降及GSH表達(dá)量下降，對(duì)海馬神經(jīng)元造成氧化損傷。
[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.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114

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