本文選題：鄰苯二甲酸-單-乙基己酯 + 自噬��； 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:鄰苯二甲酸-單-乙基己酯(Mono-(2-ethylhexyl)phthalate,MEHP)是鄰苯二甲酸二乙基己酯(Di-2-ethylhexyl phthalate,DEHP)的活性代謝產(chǎn)物。DEHP是鄰苯二甲酸酯類化合物(Phthalate esters,PAEs)中應(yīng)用最為普遍的一種人工合成的有機(jī)化合物,可以顯著提高塑料制品的可塑性和柔韌性,被廣泛地應(yīng)用到醫(yī)療用品當(dāng)中。DEHP進(jìn)入人體后很快經(jīng)肝臟和腎臟代謝成MEHP。早期的研究大部分關(guān)注MEHP對肝臟、腎臟、生殖系統(tǒng)和生長發(fā)育毒性,MEHP還具有心血管系統(tǒng)毒性,但是其分子機(jī)制尚未完全闡明。本實(shí)驗(yàn)以EA.hy926細(xì)胞為研究對象,探討MEHP作用于EA.hy926細(xì)胞后能否通過活性氧自由基(Reactive oxygen species,ROS)誘導(dǎo)自噬。方法:研究MEHP作用于EA.hy926細(xì)胞后是否通過ROS誘導(dǎo)自噬的發(fā)生,應(yīng)用MTT法(四甲基偶氮唑鹽微量酶反應(yīng)比色法)檢測不同濃度(0μM-1600μM)MEHP處理后的EA.hy926細(xì)胞存活率的變化;使用不同試劑自噬抑制劑3-甲基腺嘌呤(3-Methyladenine,3-MA)、自噬激活劑雷帕霉素、活性氧抑制劑N-乙酰半胱氨酸(NAC)、si RNA Akt1、Akt1激活劑胰島素預(yù)處理后,用不同濃度(0μM-400μM)MEHP處理EA.hy926細(xì)胞,檢測細(xì)胞存活率的變化;用透射電子顯微鏡觀察細(xì)胞自噬小體的超微結(jié)構(gòu);用Western blot法(蛋白質(zhì)免疫印跡法)檢測不同濃度(0μM-200μM)MEHP處理及NAC預(yù)處理后P62和LC3(細(xì)胞內(nèi)自噬標(biāo)記物-微管相關(guān)蛋白輕鏈3)蛋白表達(dá)水平的變化;用DCFH-DA(2’,7’-二氫二氯熒光素)檢測不同濃度(0μM-200μM)MEHP處理及NAC預(yù)處理后細(xì)胞內(nèi)ROS的含量變化;用熒光顯微鏡觀察經(jīng)JC-1染色后EA.hy926細(xì)胞內(nèi)線粒體膜電位的改變情況;用免疫熒光方法檢測不同濃度(0μM-200μM)MEHP處理后細(xì)胞內(nèi)p-Akt1水平的變化;使用不同試劑(NAC、si RNA Akt1、胰島素)預(yù)處理后,檢測細(xì)胞內(nèi)p-Akt1水平的變化;用Western blot法檢測si RNA Akt1、胰島素預(yù)處理后的LC3蛋白表達(dá)水平的變化。結(jié)果:經(jīng)過不同濃度MEHP(0μM-400μM)染毒24 h后,隨著MEHP濃度的增加EA.hy926細(xì)胞存活率顯著下降;用3-MA、NAC和胰島素干預(yù)后,細(xì)胞存活率明顯增高;用雷帕霉素、si RNA Akt1干預(yù)后,細(xì)胞存活率明顯下降。在透射電子顯微鏡下可觀察到自噬小體,且數(shù)量增加;LC3-II和P62蛋白表達(dá)隨MEHP濃度增加而增加,這說明聚集的自噬小體是由于自噬小體降解障礙引起的,用NAC預(yù)處理后,LC3-II蛋白表達(dá)量減少;細(xì)胞內(nèi)ROS水平隨MEHP劑量的升高而升高,用NAC預(yù)處理后可有效降低ROS水平;線粒體膜電位隨MEHP濃度的升高而下降,用NAC預(yù)處理后可顯著升高使線粒體膜電位;MEHP處理后細(xì)胞內(nèi)p-Akt1水平下降,用NAC、胰島素預(yù)處理后p-akt1水平升高,用si RNA Akt1干預(yù)后,p-Akt1水平顯著下降;用胰島素預(yù)處理后,LC3-II蛋白表達(dá)水平降低,而si RNA Akt1干預(yù)后則增高。結(jié)論:MEHP作用于EA.hy926細(xì)胞后,可促使細(xì)胞內(nèi)自噬小體的形成,MEHP主要依賴于ROS通過Akt1通路誘導(dǎo)自噬性細(xì)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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114

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