本文選題：環(huán)境內分泌干擾物 + 鄰苯二甲酸酯�。� 參考：《復旦大學》2014年博士論文

【摘要】：[背景]近年來,越來越多的化學物被鑒定為環(huán)境內分泌干擾物,其引起的人類多種健康效應,如神經退行性病變、癌癥、以及不孕不育等已引起人們的廣泛關注。男性不育癥(MFI)是全球生殖領域中亟待解決的重要科學問題,是常見的生殖功能障礙之一。導致男性不育的因素如遺傳、環(huán)境因素以及激素等。多項研究顯示,男性不育與環(huán)境內分泌干擾物暴露存在密切相關,內分泌干擾物能夠引起男性生殖系統損傷。隨著人們對環(huán)境化學物引起人體生殖健康關注度迅速上升,迫切需要對環(huán)境化學物引起人體或動物的內分泌系統的影響,特別是對其引起男性不育癥以及精子質量的影響進行深入研究。鄰苯二甲酸酯是一類1,2-苯二羧酸的烷基或烷基芳基酯的工業(yè)化學物,是非常重要的人工合成化合物,應用十分廣泛。鄰苯二甲酸2-乙基己酯(DEHP)是使用最為廣泛的鄰苯二甲酸酯類化合物之一,廣泛用作塑料的軟化劑和增塑劑,也是最為常見的環(huán)境內分泌干擾物。多項研究均顯示,DEHP暴露引起機體多種毒性效應,包括生殖毒性,但其引起機體生殖毒性的具體機制,特別是對男性生殖系統的影響的分子機制尚不清楚,迫切需要對其引起男性不育的具體機制進行深入研究。[目的]本研究主要從體內剛斷乳SD雄性大鼠、大鼠全胚胎培養(yǎng)以及體外睪丸細胞培養(yǎng)等三個方面系統研究DEHP引起的生殖與發(fā)育毒性及其具體分子機制,運用細胞培養(yǎng)、流式細胞術、免疫熒光以及蛋白免疫印跡等技術,系統研究DEHP引起大鼠睪丸細胞凋亡的具體信號通路以及在此過程中細胞線粒體功能、SIRT1活性以及NAD+水平的作用。1.系統研究DEHP的抗雄激素效應及其引起睪丸細胞凋亡的具體信號通路；2.觀察DEHP對大鼠全胚胎生長發(fā)育的影響。3.觀察DEHP引起多種睪丸細胞以及非睪丸細胞的細胞毒性以及對DNA復制、胞內ROS、線粒體ROS以及ATP水平等方面的影響；4.系統研究NAD+水平、SIRT1以及線粒體功能損傷在DEHP引起睪丸毒性過程中的作用；[方法]1.利用剛斷乳雄性SD大鼠模型觀察DEHP的抗雄激素效應,觀察DEHP對大鼠雄激素依賴組織如睪丸、附睪、前列腺、精囊腺、LABC以及肛門生殖器距離AGD等指標的影響；2.將大鼠睪丸、肝臟組織進行病理學檢測,HE染色觀察DEHP對大鼠睪丸、肝臟組織病理學改變,TUNEL檢測DEHP引起大鼠睪丸、肝臟組織核DNA斷裂情況；3.運用Western-blotting技術檢測DEHP引起大鼠睪丸、肝臟組織凋亡的信號通路相關蛋白的表達情況,包括caspase級聯反應蛋白如caspase-3、caspase-9、caspase-8、 PARP-1, Bcl-2家族蛋白如Bcl-XL, Bid, Bak, Bax等,系統研究DEHP引起睪丸細胞凋亡的具體信號通路；4.運用大鼠全胚胎培養(yǎng)技術觀察DEHP對大鼠全胚胎生長發(fā)育以及致畸性的影響。5.體外細胞培養(yǎng)以及DNA fiber技術檢測DEHP對大鼠睪丸間質細胞等多種細胞DNA復制的影響；6.運用流式細胞術檢測DEHP對小鼠睪丸細胞等多種細胞胞內ROS、線粒體ROS水平的影響；7.運用化學發(fā)光以及酶聯免疫法檢測DEHP對小鼠睪丸間質細胞ATP水平以及NAD+水平的影響；8.運用Western-blotting技術檢測DEHP引起大鼠睪丸、肝臟組織SIRT1、PGC-1α、乙酰化p53、p53、PARP以及氧化磷酸化OXPHOS復合物的蛋白表達變化；[結果]DEHP能夠顯著降低剛斷乳SD雄性幼鼠的雄激素依賴組織,如睪丸、附睪、前列腺、精囊腺以及LABC組織重量并引起大鼠肛門生殖器距離明顯減小,并呈現出劑量效應關系,DEHP具有顯著的抗雄激素作用。病理學檢測顯示DEHP能夠引起睪丸組織精母細胞、精子細胞數量明顯減少；TUNEL檢測結果顯示DEHP處理能夠引起大鼠睪丸組織細胞出現核DNA斷裂。利用大鼠全胚胎試驗觀察DEHP的發(fā)育毒性,結果顯示DEHP處理引起胚胎頭長、頭臀長、卵黃囊直徑等指標的明顯降低,與對照組比較具有統計學意義；DEHP處理組大鼠胚胎形態(tài)學評分明顯低于對照組,高劑量DEHP處理組大鼠胚胎出現心包腔擴大、心包積液以及后腦腫大而透明等胚胎畸形,高劑量組胚胎致畸率明顯高于對照組。因此,DEHP具有強胚胎發(fā)育毒性。運用蛋白免疫印跡技術,我們發(fā)現DEHP處理能夠引起大鼠睪丸組織中caspase-3, caspase-8以及caspase-9的蛋白表達明顯增加,同時引起B(yǎng)cl-2家族蛋白抑凋亡Bcl-XL蛋白表達明顯降低,但促凋亡蛋白Bid、Bax、Bak表達明顯升高。由此表明,DEHP引起睪丸細胞凋亡通路主要為內源性／線粒體凋亡通路。DNA fiber試驗顯示,DEHP能夠顯著抑制睪丸間質細胞CRL-2714和3T3細胞的DNA復制,DEHP導致睪丸細胞凋亡部分原因可能是其引起DNA復制叉停止,導致DNA鏈斷裂而引起。體外試驗結果顯示,DEHP處理引起多種睪丸細胞以及非睪丸細胞3T3細胞活性氧ROS以及線粒體ROS顯著升高,其中小鼠睪丸間質細胞CRL-2714以及3T3成纖維細胞對DEHP較為敏感。DEHP處理能夠顯著降低小鼠睪丸間質細胞NAD+水平。我們對與NAD+密切相關的去乙�；窼IRT1蛋白表達情況進行分析。結果顯示,DEHP處理能夠顯著降低睪丸組織中SIRT1以及PGC-la蛋白表達。DEHP處理引起大鼠睪丸組織p53表達以及去乙酰化p53蛋白的表達明顯升高。利用化學發(fā)光法檢測DEHP對小鼠睪丸間質細胞ATP水平的影響,結果顯示DEHP能夠顯著降低細胞ATP水平。對DEHP對睪丸組織中氧化磷酸化產物復合物蛋白表達情況進行研究,結果顯示DEHP能夠降低氧化磷酸化產物Ⅱ,Ⅲ,Ⅳ和Ⅴ的表達,與對照組比較具有統計學意義。綜上,DEHP引起睪丸細胞凋亡以及細胞線粒體功能損傷可能與其引起多聚ADP核糖聚合反應,導致NAD+過度消耗,進而損傷SIRT1活性有關。SIRT1活性降低、線粒體功能損傷和DNA復制抑制可能參與DEHP引起的睪丸毒性過程。本研究首次闡述了DEHP引起睪丸毒性可能的作用機制,明確SIRT1、線粒體功能以及DNA復制在DEHP引起睪丸毒性過程中的作用。DEHP處理引起復制叉停止,抑制DNA的復制,激活PARP,進而引起NAD+水平降低,SIRT1活性降低,引起睪丸細胞線粒體功能損傷以及線粒體通路細胞凋亡。肝臟亦是DEHP毒性的重要靶器官之一,我們對DEHP引起的肝臟毒性進行了深入研究。病理學檢測發(fā)現,DEHP處理引起大鼠肝細胞間隙增大,肝細胞濁脹,部分細胞壞死并伴有脂肪性病變。TUNEL檢測顯示,DEHP引起明顯的肝臟細胞核DNA斷裂。DEHP處理引起大鼠肝臟組織caspase-8, caspase-9, caspase-3以及PARP蛋白表達明顯增加,但DEHP處理僅引起大鼠肝臟組織Bcl-2家族蛋白的引起促凋亡蛋白Bid, Bak, Bax輕度變化,這表明DEHP引起肝臟細胞凋亡途徑并非主要通過內源性凋亡通路。同時,我們發(fā)現DEHP處理引起肝臟組織Bcl-XL蛋白表達明顯增加,這表明可能是DEHP引起二次細胞保護反應的影響。DEHP處理未引起大鼠肝臟組織起氧化磷酸化復合物Ⅱ、Ⅲ、Ⅳ和Ⅴ表達的明顯變化。[結論]1. DEHP具有抗雄激素作用,DEHP處理能夠引起剛斷乳雄性SD幼鼠的生殖系統損傷；2. DEHP具有強胚胎發(fā)育毒性。3. DEHP引起睪丸細胞凋亡可能與抑制DNA復制引起的DNA損傷有關；4. DEHP引起睪丸細胞凋亡通路主要是內源性/線粒體凋亡通路；5. DEHP引起線粒體功能損傷與PARP活化以及SIRT1去乙�；磻獙е翹AD+過度消耗有關；6. SIRT1可能在男性生殖過程中具有重要作用；
[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.

【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R114

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