邻苯二甲酸2-乙基己酯对雄性幼鼠生殖毒性及机制研究

发布时间：2018-05-04 19:27

本文选题：环境内分泌干扰物 + 邻苯二甲酸酯　；参考：《复旦大学》2014年博士论文

【摘要】：[背景]近年来,越来越多的化学物被鉴定为环境内分泌干扰物,其引起的人类多种健康效应,如神经退行性病变、癌症、以及不孕不育等已引起人们的广泛关注。男性不育症(MFI)是全球生殖领域中亟待解决的重要科学问题,是常见的生殖功能障碍之一。导致男性不育的因素如遗传、环境因素以及激素等。多项研究显示,男性不育与环境内分泌干扰物暴露存在密切相关,内分泌干扰物能够引起男性生殖系统损伤。随着人们对环境化学物引起人体生殖健康关注度迅速上升,迫切需要对环境化学物引起人体或动物的内分泌系统的影响,特别是对其引起男性不育症以及精子质量的影响进行深入研究。邻苯二甲酸酯是一类1,2-苯二羧酸的烷基或烷基芳基酯的工业化学物,是非常重要的人工合成化合物,应用十分广泛。邻苯二甲酸2-乙基己酯(DEHP)是使用最为广泛的邻苯二甲酸酯类化合物之一,广泛用作塑料的软化剂和增塑剂,也是最为常见的环境内分泌干扰物。多项研究均显示,DEHP暴露引起机体多种毒性效应,包括生殖毒性,但其引起机体生殖毒性的具体机制,特别是对男性生殖系统的影响的分子机制尚不清楚,迫切需要对其引起男性不育的具体机制进行深入研究。[目的]本研究主要从体内刚断乳SD雄性大鼠、大鼠全胚胎培养以及体外睾丸细胞培养等三个方面系统研究DEHP引起的生殖与发育毒性及其具体分子机制,运用细胞培养、流式细胞术、免疫荧光以及蛋白免疫印迹等技术,系统研究DEHP引起大鼠睾丸细胞凋亡的具体信号通路以及在此过程中细胞线粒体功能、SIRT1活性以及NAD+水平的作用。1.系统研究DEHP的抗雄激素效应及其引起睾丸细胞凋亡的具体信号通路；2.观察DEHP对大鼠全胚胎生长发育的影响。3.观察DEHP引起多种睾丸细胞以及非睾丸细胞的细胞毒性以及对DNA复制、胞内ROS、线粒体ROS以及ATP水平等方面的影响；4.系统研究NAD+水平、SIRT1以及线粒体功能损伤在DEHP引起睾丸毒性过程中的作用；[方法]1.利用刚断乳雄性SD大鼠模型观察DEHP的抗雄激素效应,观察DEHP对大鼠雄激素依赖组织如睾丸、附睾、前列腺、精囊腺、LABC以及肛门生殖器距离AGD等指标的影响；2.将大鼠睾丸、肝脏组织进行病理学检测,HE染色观察DEHP对大鼠睾丸、肝脏组织病理学改变,TUNEL检测DEHP引起大鼠睾丸、肝脏组织核DNA断裂情况；3.运用Western-blotting技术检测DEHP引起大鼠睾丸、肝脏组织凋亡的信号通路相关蛋白的表达情况,包括caspase级联反应蛋白如caspase-3、caspase-9、caspase-8、 PARP-1, Bcl-2家族蛋白如Bcl-XL, Bid, Bak, Bax等,系统研究DEHP引起睾丸细胞凋亡的具体信号通路；4.运用大鼠全胚胎培养技术观察DEHP对大鼠全胚胎生长发育以及致畸性的影响。5.体外细胞培养以及DNA fiber技术检测DEHP对大鼠睾丸间质细胞等多种细胞DNA复制的影响；6.运用流式细胞术检测DEHP对小鼠睾丸细胞等多种细胞胞内ROS、线粒体ROS水平的影响；7.运用化学发光以及酶联免疫法检测DEHP对小鼠睾丸间质细胞ATP水平以及NAD+水平的影响；8.运用Western-blotting技术检测DEHP引起大鼠睾丸、肝脏组织SIRT1、PGC-1α、乙酰化p53、p53、PARP以及氧化磷酸化OXPHOS复合物的蛋白表达变化；[结果]DEHP能够显著降低刚断乳SD雄性幼鼠的雄激素依赖组织,如睾丸、附睾、前列腺、精囊腺以及LABC组织重量并引起大鼠肛门生殖器距离明显减小,并呈现出剂量效应关系,DEHP具有显著的抗雄激素作用。病理学检测显示DEHP能够引起睾丸组织精母细胞、精子细胞数量明显减少；TUNEL检测结果显示DEHP处理能够引起大鼠睾丸组织细胞出现核DNA断裂。利用大鼠全胚胎试验观察DEHP的发育毒性,结果显示DEHP处理引起胚胎头长、头臀长、卵黄囊直径等指标的明显降低,与对照组比较具有统计学意义；DEHP处理组大鼠胚胎形态学评分明显低于对照组,高剂量DEHP处理组大鼠胚胎出现心包腔扩大、心包积液以及后脑肿大而透明等胚胎畸形,高剂量组胚胎致畸率明显高于对照组。因此,DEHP具有强胚胎发育毒性。运用蛋白免疫印迹技术,我们发现DEHP处理能够引起大鼠睾丸组织中caspase-3, caspase-8以及caspase-9的蛋白表达明显增加,同时引起Bcl-2家族蛋白抑凋亡Bcl-XL蛋白表达明显降低,但促凋亡蛋白Bid、Bax、Bak表达明显升高。由此表明,DEHP引起睾丸细胞凋亡通路主要为内源性／线粒体凋亡通路。DNA fiber试验显示,DEHP能够显著抑制睾丸间质细胞CRL-2714和3T3细胞的DNA复制,DEHP导致睾丸细胞凋亡部分原因可能是其引起DNA复制叉停止,导致DNA链断裂而引起。体外试验结果显示,DEHP处理引起多种睾丸细胞以及非睾丸细胞3T3细胞活性氧ROS以及线粒体ROS显著升高,其中小鼠睾丸间质细胞CRL-2714以及3T3成纤维细胞对DEHP较为敏感。DEHP处理能够显著降低小鼠睾丸间质细胞NAD+水平。我们对与NAD+密切相关的去乙酰化酶SIRT1蛋白表达情况进行分析。结果显示,DEHP处理能够显著降低睾丸组织中SIRT1以及PGC-la蛋白表达。DEHP处理引起大鼠睾丸组织p53表达以及去乙酰化p53蛋白的表达明显升高。利用化学发光法检测DEHP对小鼠睾丸间质细胞ATP水平的影响,结果显示DEHP能够显著降低细胞ATP水平。对DEHP对睾丸组织中氧化磷酸化产物复合物蛋白表达情况进行研究,结果显示DEHP能够降低氧化磷酸化产物Ⅱ,Ⅲ,Ⅳ和Ⅴ的表达,与对照组比较具有统计学意义。综上,DEHP引起睾丸细胞凋亡以及细胞线粒体功能损伤可能与其引起多聚ADP核糖聚合反应,导致NAD+过度消耗,进而损伤SIRT1活性有关。SIRT1活性降低、线粒体功能损伤和DNA复制抑制可能参与DEHP引起的睾丸毒性过程。本研究首次阐述了DEHP引起睾丸毒性可能的作用机制,明确SIRT1、线粒体功能以及DNA复制在DEHP引起睾丸毒性过程中的作用。DEHP处理引起复制叉停止,抑制DNA的复制,激活PARP,进而引起NAD+水平降低,SIRT1活性降低,引起睾丸细胞线粒体功能损伤以及线粒体通路细胞凋亡。肝脏亦是DEHP毒性的重要靶器官之一,我们对DEHP引起的肝脏毒性进行了深入研究。病理学检测发现,DEHP处理引起大鼠肝细胞间隙增大,肝细胞浊胀,部分细胞坏死并伴有脂肪性病变。TUNEL检测显示,DEHP引起明显的肝脏细胞核DNA断裂。DEHP处理引起大鼠肝脏组织caspase-8, caspase-9, caspase-3以及PARP蛋白表达明显增加,但DEHP处理仅引起大鼠肝脏组织Bcl-2家族蛋白的引起促凋亡蛋白Bid, Bak, Bax轻度变化,这表明DEHP引起肝脏细胞凋亡途径并非主要通过内源性凋亡通路。同时,我们发现DEHP处理引起肝脏组织Bcl-XL蛋白表达明显增加,这表明可能是DEHP引起二次细胞保护反应的影响。DEHP处理未引起大鼠肝脏组织起氧化磷酸化复合物Ⅱ、Ⅲ、Ⅳ和Ⅴ表达的明显变化。[结论]1. DEHP具有抗雄激素作用,DEHP处理能够引起刚断乳雄性SD幼鼠的生殖系统损伤；2. DEHP具有强胚胎发育毒性。3. DEHP引起睾丸细胞凋亡可能与抑制DNA复制引起的DNA损伤有关；4. DEHP引起睾丸细胞凋亡通路主要是内源性/线粒体凋亡通路；5. DEHP引起线粒体功能损伤与PARP活化以及SIRT1去乙酰化反应导致NAD+过度消耗有关；6. SIRT1可能在男性生殖过程中具有重要作用；
[Abstract]:[background] more and more chemicals have been identified as environmental endocrine disruptors in recent years. Many human health effects, such as neurodegenerative diseases, cancer, and infertility, have aroused widespread concern. Male infertility (MFI) is an important scientific problem to be solved in the global reproductive field. It is a common reproductive work. One of the obstacles. Factors that cause male infertility such as heredity, environmental factors and hormones. A number of studies have shown that male infertility is closely related to environmental endocrine disruptors exposure, and endocrine disruptors can cause male reproductive system damage. The impact of environmental chemicals on the endocrine system of the human body or animal, especially the effects on male infertility and sperm quality, is studied. Phthalate is an industrial chemical of alkyl or alkyl aryl ester of a class of 1,2- benzene two carboxylic acid. It is a very important synthetic compound and is used very well. 2- ethylhexyl phthalate (DEHP) is one of the most widely used phthalic acid esters. It is widely used as a plasticizer and plasticizer. It is also the most common environmental endocrine disruptor. A number of studies have shown that DEHP exposure causes many toxic effects, including reproductive toxicity, but it causes the reproduction of the body. The specific mechanism of toxicity, especially the molecular mechanism of the effect on male reproductive system, is not clear, and it is urgent to study the specific mechanism of male infertility. [Objective] this study is to systematically study the three aspects of DEHP in SD male rats, the whole embryo culture and the culture of testicular cells in vitro. Reproductive and developmental toxicity and its specific molecular mechanism, cell culture, flow cytometry, immunofluorescence and protein immunoblotting were used to systematically study the specific signaling pathway of DEHP induced apoptosis in rat testicular cells, and the function of mitochondria, SIRT1 activity and the role of NAD+ in the process of DE in the study of DE. The anti androgen effect of HP and the specific signaling pathway to induce apoptosis of testicular cells; 2. the effect of DEHP on the growth and development of the whole embryo in rats;.3. observed the cytotoxicity of DEHP in many testis and non testicular cells, and on the effects on DNA replication, intracellular ROS, mitochondrial ROS and ATP levels; and 4. systematic study of NAD+ water The effect of SIRT1 and mitochondrial function damage on the toxicity of DEHP in testis was observed. [method]1. observed the anti androgen effect of DEHP in the male SD rat model with rigid weaning, and observed the effects of DEHP on the androgen dependent tissue such as testicles, epididymis, prostate, seminal vesicle, LABC and the distance of the anus genitals to AGD. 2. Pathological examination of rat testis and liver tissue, HE staining was used to observe the pathological changes of rat testis and liver tissues by DEHP. TUNEL detection of DEHP caused the rupture of DNA in rat testis and liver tissue; 3. the expression of signaling pathway related proteins in rat testis and liver tissue induced by DEHP was detected by Western-blotting technology, including the expression of signal pathway related proteins in rat liver tissue. Caspase cascade reaction proteins such as Caspase-3, caspase-9, Caspase-8, PARP-1, Bcl-2 family proteins such as Bcl-XL, Bid, Bak, Bax, etc., systematically study the specific signaling pathway of apoptosis in testicular cells by DEHP, and 4. to observe the effect of DEHP on the growth and teratogenicity of the whole embryo and the teratogenicity of rats by the whole embryo culture technique. The effect of DEHP on DNA replication in rat Leydig cells, and the effect of DEHP on DNA replication in rat Leydig cells; 6. the effect of DEHP on the intracellular ROS and mitochondrial ROS in mice testis cells was detected by flow cytometry; and 7. using chemiluminescence and ELISA to detect the ATP level and NAD+ of DEHP in mice testis stromal cells. The effects of Western-blotting on the expression of DEHP in rat testis, SIRT1, PGC-1 a, acetylated p53, p53, PARP, and oxidative phosphorylation of OXPHOS complex were detected by Western-blotting technique. [results]DEHP can significantly reduce the androgen dependence of newborn SD male young rats, such as testis, epididymis, prostate, seminal vesicle. As well as the weight of LABC tissue, the distance of the anus genitals in rats decreased significantly and showed a dose effect relationship. DEHP had a significant anti androgen effect. Pathological examination showed that DEHP could cause spermatocyte in testis tissue and the number of spermatocyte decreased significantly; TUNEL detection showed that DEHP treatment could cause testis tissue in rats. The development toxicity of DEHP was observed by the whole embryo test in rats. The results showed that the DEHP treatment caused the head length, the length of the head and the hip, the yolk sac diameter and so on, which was significantly lower than the control group. The morphology score of the DEHP treatment group was significantly lower than that of the control group, and the high dose DEHP treatment group was larger than the control group. In the rat embryo, the pericardial cavity enlargement, pericardial effusion, and the swelling of the posterior brain and transparent and other embryonic deformities. The rate of teratogenesis in the high dose group is significantly higher than that of the control group. Therefore, DEHP has a strong embryonic developmental toxicity. Using protein immunoblotting technique, we found that DEHP treatment could cause Caspase-3, Caspase-8 and caspase-9 in the rat testis tissue. The expression of protein expression increased obviously, and the expression of Bcl-XL protein expression in Bcl-2 family protein decreased obviously, but the expression of apoptotic protein Bid, Bax and Bak increased obviously. Thus, DEHP induced apoptosis pathway of testis cells mainly endogenous / mitochondrial apoptotic pathway.DNA fiber test display, DEHP could significantly inhibit the CRL-2 of Leydig cell CRL-2. The DNA replication of 714 and 3T3 cells, the cause of DEHP induced apoptosis in testicular cells may be caused by the stop of the DNA replication fork and cause the disruption of the DNA chain. In vitro results showed that DEHP treatment caused a variety of testicular cells and non testicular cells 3T3 cells active oxygen ROS and mitochondrial ROS significantly increased, including mouse Leydig cells CRL-. 2714 and the more sensitive.DEHP treatment of 3T3 fibroblasts to DEHP could significantly reduce the level of NAD+ in mouse Leydig cells. We analyzed the expression of deacetylase SIRT1 protein closely related to NAD+. The results showed that DEHP treatment could significantly reduce SIRT1 and PGC-la protein expression of.DEHP in testis. The expression of p53 and the expression of deacetylation of p53 protein in rat testis were significantly increased. The effects of DEHP on the ATP level of Leydig cells in mouse testis were detected by chemiluminescence. The results showed that DEHP could significantly reduce the level of ATP in the cells. The expression of the complex protein of the oxidative phosphorylated product in the testicular tissue was studied by DEHP, and the results showed D. EHP can reduce the expression of oxidative phosphorylation Products II, III, IV and V, which is statistically significant compared with the control group. To sum up, DEHP induces apoptosis and mitochondrial function damage in testicular cells and may lead to the polymerization of poly ADP ribose, resulting in the excessive consumption of NAD+ and the decrease of the activity of SIRT1 on the activity of.SIRT1. Functional injury and inhibition of DNA replication may be involved in the toxic process of testicle induced by DEHP. This study first described the possible mechanism of DEHP induced toxicity of testicular toxicity, clearly SIRT1, mitochondrial function, and the role of DNA replication in DEHP induced testicular toxicity..DEHP treatment caused the stop of replication forks, inhibition of DNA replication, activation of PARP, and further induction The level of NAD+ decreased and the activity of SIRT1 decreased, causing mitochondrial function damage and mitochondrial apoptosis. The liver is one of the important target organs of DEHP toxicity. We have studied the liver toxicity caused by DEHP. Pathological examination found that DEHP treatment caused the enlargement of the hepatic cell space, the turbid liver cell and the part of the liver. Cell necrosis and.TUNEL detection of fatty lesions showed that DEHP induced the obvious DNA break of the liver nucleus by.DEHP treatment. The expression of caspase-8, caspase-9, caspase-3 and PARP protein increased significantly in rat liver tissue, but DEHP treatment only caused the Bcl-2 family protein of the rat liver to induce apoptotic protein Bid, Bak. Degree change, which indicates that the DEHP induced apoptosis pathway is not mainly through the endogenous apoptotic pathway. At the same time, we found that the expression of Bcl-XL protein in the liver tissue increased significantly by DEHP treatment, suggesting that it may be the effect of DEHP on the two cell protective response to.DEHP treatment that did not induce the oxidative phosphorylation complex of rat liver tissue. [conclusion]1. DEHP has anti androgen effect, and DEHP treatment can induce reproductive system damage in newborn male SD young rats; 2. DEHP with strong embryonic developmental toxicity.3. DEHP causes apoptosis of testicular cells may be related to the inhibition of DNA injury caused by DNA replication; 4. DEHP induces apoptosis of testicular cells. The main pathway is endogenous / mitochondrial apoptosis pathway, and 5. DEHP induced mitochondrial dysfunction is associated with PARP activation and SIRT1 deacetylation resulting in excessive consumption of NAD+; 6. SIRT1 may play an important role in male reproductive process.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114

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