【摘要】：鄰苯二甲酸酯(phthalate esters, PEs),是一類普遍使用的化學(xué)工業(yè)品,主要用作增塑劑,其中鄰苯二甲酸二乙基己基酯(Di-2-ethylhexyl phthalate, DEHP)和鄰苯二甲酸二丁酯(Dibutyl phthalate, DBP)是使用較為廣泛的增塑劑。鄰苯二甲酸-單-乙基己基酯(Mono-2-ethylhexyl phthalate, MEHP)和鄰苯二甲酸單丁酯(Mono-n-butyl phthalate, MBP)分別是DEHP和DBP的主要代謝物。使用過程中,鄰苯二甲酸酯會通過各種途徑進(jìn)入環(huán)境中,人類可經(jīng)由食品、水和土壤接觸鄰苯二甲酸酯�，F(xiàn)已發(fā)現(xiàn)鄰苯二甲酸酯具有很強(qiáng)的生殖和發(fā)育毒性,能損害生物的生育力。然而,目前我們對于MEHP和MBP對植入前胚胎發(fā)育的影響仍知之甚少。 為了研究MEHP對植入前胚胎發(fā)育的影響,我們用0,10-5,10-4,10-3M MEHP處理受精卵,發(fā)現(xiàn)10-3M MEHP會引起小鼠胚胎發(fā)生2-細(xì)胞阻滯。10-3M MEHP暴露24h后,小鼠胚胎細(xì)胞內(nèi)活性氧水平顯著升高。然而,抗氧化物(CAT和SOD)可以降低胚胎細(xì)胞中活性氧的水平,提高胚胎的存活率,但是沒有恢復(fù)發(fā)生2-細(xì)胞阻滯胚胎的發(fā)育。進(jìn)一步的實(shí)驗(yàn)結(jié)果表明,MEHP暴露后,只有死亡胚胎的凋亡水平升高了,但活的2-細(xì)胞阻滯的胚胎凋亡水平?jīng)]有明顯變化。這些結(jié)果提示ROS水平的升高和胚胎細(xì)胞凋亡并不是發(fā)生2-細(xì)胞發(fā)育胎阻滯的主要原因。通過分析胚胎基因組激活(embryonic genome activation, EGA)的關(guān)鍵基因(Hsc70, MuERV-L, Hsp70.1, eIF-1A和Zscan4)以及母源效應(yīng)基因(OCT4和SOX2),我們發(fā)現(xiàn)胚胎2-細(xì)胞到4-細(xì)胞階段,MEHP暴露顯著降低了基因Hsc70、MuERV-L和SOX2的表達(dá)水平,提高了Hsp70.1、eIF-1A、Zscan4和OCT4的表達(dá)水平。添加抗氧化物(CAT和SOD)沒能改變這些基因的表達(dá)趨勢。由此可見,MEHP-誘導(dǎo)的胚胎發(fā)育阻滯是由母體向胚胎轉(zhuǎn)換異常介導(dǎo)的,而不是氧化應(yīng)激和細(xì)胞凋亡。 為了研究MBP對植入前胚胎發(fā)育的影響,我們用0,10-5,10-4,10-3M MBP處理受精卵,發(fā)現(xiàn)10-3和10-4M MBP暴露96h,囊胚的數(shù)量均顯著降低,當(dāng)延遲培養(yǎng)24h時,即120h,10-3M MBP暴露組桑椹胚繼續(xù)發(fā)育到囊胚的胚胎數(shù)量顯著高于對照組,但96h和120h囊胚的總數(shù)仍顯著低于對照組,并且10-3M MBP暴露顯著降低了孵化囊胚的數(shù)量,這些結(jié)果顯示了10-3M MBP會損害小鼠植入前胚胎的發(fā)育潛能,10-4M MBP暴露只是延緩了胚胎的發(fā)育進(jìn)程。10-3M MBP暴露48h后,胚胎細(xì)胞內(nèi)ROS水平顯著升高,并通過促進(jìn)線粒體釋放細(xì)胞色素c提高了凋亡水平。免疫熒光染色分析顯示MBP處理以劑量依賴(10-5,10-4,10-3M)的方式降低了DNA甲基化水平。這些結(jié)果顯示MBP可能通過誘導(dǎo)氧化應(yīng)激、細(xì)胞凋亡或抑制DNA甲基化降低胚胎的發(fā)育潛能。 總之,本研究首次揭示了PEs暴露能對植入前胚胎發(fā)育造成損害。MEHP能夠引起胚胎2-細(xì)胞發(fā)育阻滯,其主要是由于EGA啟動失敗和母源效應(yīng)基因表達(dá)異常引起,與ROS和凋亡水平無顯著相關(guān)性。MBP能夠降低植入前胚胎的發(fā)育潛能,其主要與ROS和細(xì)胞凋亡相關(guān),還可能與DNA甲基化水平的降低有關(guān)。
[Abstract]:Phthalate (phthalate esters, PEs), is a widely used chemical industry, mainly used as plasticizer, in which diethylhexyl phthalate (Di-2-ethylhexyl phthalate, DEHP) and dibutyl phthalate (Dibutyl phthalate,) DBP) is a widely used plasticizer. Monoethyl hexyl phthalate (Mono-2-ethylhexyl phthalate, MEHP) and monobutyl phthalate (Mono-n-butyl phthalate, MBP) are the main metabolites of DEHP and DBP, respectively. During use, phthalates enter the environment through various channels, and humans can access phthalates through food, water and soil. It has been found that phthalates have strong reproductive and developmental toxicity, which can damage the fertility of organisms. However, little is known about the effects of MEHP and MBP on the development of preimplantation embryos. In order to study the effect of MEHP on preimplantation embryo development, we treated fertilized eggs with 0, 10 ~ 5, 10 ~ 4, 10 ~ 3 M MEHP, and found that 10 ~ (3) M MEHP induced 2-cell arrest in mouse embryos. 24 hours after exposure to 10-3 M MEHP, we found that 10-3 M MEHP induced 2-cell block in mouse embryos. The level of reactive oxygen species (Ros) in mouse embryo cells increased significantly. However, antioxidants (CAT and SOD) decreased the level of reactive oxygen species (Ros) in embryonic cells and increased the survival rate of embryos, but did not resume 2-cell block of embryo development. The further results showed that only the apoptotic level of dead embryos increased after MEHP exposure, but there was no significant change in the apoptotic level of live 2-cell arrest embryos. These results suggest that the increase of ROS level and embryonic cell apoptosis are not the main causes of 2-cell fetal block. By analyzing the key genes that activate (embryonic genome activation, EGA) in the embryonic genome (Hsc70, MuERV-L, Hsp70.1, eIF-1A and Zscan4) and maternal effectors (OCT4 and SOX2), we found that embryonic 2-cell to 4-cell stage. MEHP exposure significantly decreased the expression level of gene Hsc70,MuERV-L and SOX2, and increased the expression level of Hsp70.1,eIF-1A,Zscan4 and OCT4. The addition of antioxidants (CAT and SOD) did not change the expression trend of these genes. It can be seen that MEHP- induced embryonic development block is mediated by abnormal transformation from mother to embryo, not oxidative stress and apoptosis. In order to study the effect of MBP on the development of preimplantation embryos, we treated the fertilized eggs with 0, 10, 5, 10, 3 M MBP. It was found that the number of blastocysts decreased significantly after exposure to 10, 10 and 4 M MBP for 96 hours, and the number of blastocysts decreased significantly at 24 hours of delayed culture, that is, 120 hours. The number of blastocysts continued to develop into blastocysts in the group exposed to 10 m MBP was significantly higher than that in the control group, but the total number of blastocysts at 96 h and 120 h was still significantly lower than that in the control group, and the number of hatched blastocysts was significantly decreased after exposure to 10 m MBP. These results showed that 10 m MBP could damage the developmental potential of mouse preimplantation embryos, and 10 m MBP exposure only delayed the development of embryos. 48 h after exposure to 10-3 M MBP, the level of ROS in embryonic cells increased significantly. The level of apoptosis was increased by promoting the release of cytochrome c from mitochondria. Immunofluorescence staining showed that MBP treatment reduced the level of DNA methylation in a dose-dependent manner (10 ~ 5, 10 ~ 4, 10 ~ 3 M). These results suggest that MBP may reduce the developmental potential of embryos by inducing oxidative stress, apoptosis or inhibition of DNA methylation. In conclusion, this study revealed for the first time that PEs exposure can cause damage to preimplantation embryo development. MEHP can cause embryonic 2-cell development block, mainly due to the failure of EGA initiation and abnormal expression of maternal effector genes. MBP can decrease the developmental potential of preimplantation embryos, which is mainly related to ROS and apoptosis, and may also be related to the decrease of DNA methylation level.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R114

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相關(guān)期刊論文 前1條

1 王立鑫;楊旭;;鄰苯二甲酸酯毒性及健康效應(yīng)研究進(jìn)展[J];環(huán)境與健康雜志;2010年03期

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本文編號：2449316