本文關(guān)鍵詞： 雙酚A β細(xì)胞 胰十二指腸同源盒1 組蛋白修飾 DNA甲基化 血糖穩(wěn)態(tài) 雙酚A 學(xué)習(xí)記憶 焦慮行為 皮質(zhì)酮 雌激素受體 DNA甲基化　出處：《華中科技大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：英國科學(xué)家David J. P. Baker在1995年首次提出了“成人疾病的胎兒起源”(Fetal Origins of Adult Disease,FOAD)假說,后來逐漸演變成了如今的“健康和疾病的發(fā)育起源”(Developmental Origins of Health and Disease, DOHaD)假說。該假說認(rèn)為人類在早期發(fā)育過程中(包括胎兒、嬰兒、兒童時期)經(jīng)歷不利因素,組織器官在結(jié)構(gòu)和功能上會發(fā)生永久性或程序性改變,將會影響成年期糖尿病、代謝綜合征、心血管疾病、精神行為異常等慢性非傳染性疾病的發(fā)生發(fā)展。內(nèi)分泌干擾物,作為廣泛存在的環(huán)境污染物,其發(fā)育期暴露對器官系統(tǒng)的發(fā)育毒性及與成年期疾病易感性的關(guān)系已引起越來越多的關(guān)注。雙酚A (BPA),是目前世界范圍內(nèi)制造和使用量最多的環(huán)境內(nèi)分泌干擾物之一。作為單體分子,BPA主要用于生產(chǎn)制造聚碳酸酯和環(huán)氧樹脂等可用于食品和飲料包裝的高分子材料。BPA暴露主要經(jīng)由食品包裝和飲料容器中滲入到食物中從而經(jīng)口攝入,其他如皮膚接觸和呼吸攝入等也是重要暴露途徑。而且,研究表明BPA可以經(jīng)過胎盤和母乳傳遞給胎兒和新生兒,對人類健康形成嚴(yán)重威脅。動物研究和人群流行病學(xué)研究都表明發(fā)育期暴露于BPA可改變成年期神經(jīng)行為包括學(xué)習(xí)／記憶和情緒調(diào)節(jié),但是其潛在的機(jī)制尚不明確。前期研究同樣表明發(fā)育期BPA暴露可增加成年期糖尿病易感性,且機(jī)制同樣尚不明確。本研究首先從胰腺發(fā)育的角度探討大鼠發(fā)育期暴露于BPA致成年期糖代謝紊亂的可能途徑,及其中潛在的表觀遺傳機(jī)制。本研究接著探討發(fā)育期暴露于BPA對成年期大鼠學(xué)習(xí)記憶和焦慮行為的影響,以及對控制相應(yīng)行為的大腦區(qū)域內(nèi)相關(guān)基因表達(dá)的影響,并探討潛在的表觀遺傳機(jī)制。第一部分表觀遺傳介導(dǎo)BPA發(fā)育期暴露干擾大鼠胰腺發(fā)育并誘導(dǎo)成年期糖代謝紊亂目的：探討發(fā)育期暴露于低劑量BPA對胰腺內(nèi)p細(xì)胞早期發(fā)育的影響,及與此有關(guān)的控制胰腺發(fā)育的關(guān)鍵基因的表達(dá)。在此基礎(chǔ)上,探討B(tài)PA暴露引起的早期胰腺發(fā)育受損潛在的表觀遺傳機(jī)制及對成年期糖代謝的影響。方法：將受孕的Wistar大鼠按體重匹配后隨機(jī)分成對照組和BPA暴露組。從妊娠第一天開始,每天經(jīng)口給予暴露組孕鼠10μg/kg體重的BPA,對照組給予相應(yīng)的溶劑對照玉米油。大鼠出生后,利用免疫組化和定量PCR檢測胰腺β細(xì)胞發(fā)育及關(guān)鍵調(diào)控基因的表達(dá)；在此基礎(chǔ)上,分離胚胎15.5天胰腺觀察胰腺發(fā)育關(guān)鍵基因的表達(dá)；大鼠出生后至成年,利用葡萄糖耐量試驗和胰島素耐量試驗,順序觀察糖代謝情況；從胚胎期至成年,利用染色質(zhì)免疫共沉淀和質(zhì)譜法,持續(xù)測定胰腺發(fā)育過程中關(guān)鍵調(diào)控基因的組蛋白修飾和DNA甲基化改變。結(jié)果：胚胎期暴露于10μg/kg/d BPA可降低出生時仔鼠胰腺p細(xì)胞質(zhì)量(Beta-cell mass, BCM),并降低胰腺發(fā)育關(guān)鍵調(diào)控基因胰十二指腸同源盒1(pancreatic duodenal homeobox-1, Pdx1)的表達(dá)。進(jìn)一步研究發(fā)現(xiàn),BPA對胰腺Pdxl表達(dá)的影響在妊娠15.5天既己出現(xiàn),表現(xiàn)為胰腺內(nèi)PDX1陽性細(xì)胞分?jǐn)?shù)降低。BPA暴露在胚胎期顯著改變Pdxl啟動子區(qū)域的組蛋白修飾,使之表達(dá)失活；主要表現(xiàn)為組蛋白H3和H4乙酰化降低,組蛋白H3第4位賴氨酸三甲基化降低和9位賴氨酸雙甲基化升高；且此種失活調(diào)節(jié)趨勢可隨著胎兒生長發(fā)育進(jìn)行性加深,直至成年期；然而,本研究中Pdxl基因啟動子區(qū)域DNA甲基化檢測未見明顯改變；母體BPA暴露最終使成年期子代發(fā)生糖代謝紊亂。結(jié)論：發(fā)育期暴露于BPA可顯著改變胰腺發(fā)育關(guān)鍵調(diào)控基因Pdxl啟動子區(qū)域的組蛋白修飾,使之向失活狀態(tài)轉(zhuǎn)變,從而影響早期胰腺p細(xì)胞發(fā)育,最終在成年期引起糖代謝紊亂。第二部分BPA發(fā)育期暴露損害大鼠學(xué)習(xí)記憶能力和海馬區(qū)雌激素受體α的DNA甲基化目的：探討發(fā)育期暴露于雙酚A(BPA)對子代大鼠學(xué)習(xí)記憶行為和情緒調(diào)節(jié)及探索行為的影響,并探討其中的表觀遺傳學(xué)機(jī)制。方法：將受孕的Sprague-Dawley (SD)大鼠按體重匹配后隨機(jī)分成對照組和BPA暴露組。從妊娠第一天開始至出生后21天斷乳,每天灌胃給予暴露組大鼠40μg/kg體重的BPA,對照組給予相應(yīng)的溶劑對照玉米油。子代大鼠出生后60天,利用Morris水迷宮測定大鼠的學(xué)習(xí)記憶能力；子代大鼠出生后85天,利用高架十字迷宮測定大鼠的焦慮和探索行為；應(yīng)用ELISA方法測定大鼠血清中的皮質(zhì)酮濃度,同時應(yīng)用定量PCR法測定子代大鼠海馬組織中雌激素受體α和雌激素受體β的表達(dá)水平；最后測定子代大鼠海馬中雌激素受體α的基因啟動子區(qū)域甲基化狀態(tài),探討B(tài)PA暴露影響子代大鼠學(xué)習(xí)記憶行為發(fā)育的表觀遺傳學(xué)機(jī)制。結(jié)果：水迷宮實驗中,發(fā)育期暴露于BPA首先顯著延長了雌雄子代大鼠在獲得性訓(xùn)練中找到潛伏平臺的時間,其次在探查訓(xùn)練中降低了子代大鼠出現(xiàn)在目標(biāo)象限的時間。高架十字迷宮實驗中,發(fā)育期暴露于BPA沒有在子代大鼠中產(chǎn)生有統(tǒng)計學(xué)意義的行為改變；但是,BPA暴露子代大鼠血清中的皮質(zhì)酮濃度相比于對照組子代大鼠顯著升高,且此種改變呈現(xiàn)出性別特異性。相對于對照組,發(fā)育期暴露于BPA顯著降低了子代大鼠海馬雌激素受體α的表達(dá)水平,而雌激素受體β沒有明顯改變。發(fā)育期暴露于BPA還顯著升高了子代大鼠海馬雌激素受體α基因啟動子區(qū)域的DNA甲基化。結(jié)論：發(fā)育期暴露于BPA使子代大鼠學(xué)習(xí)記憶能力受損,此種受損與子代大鼠海馬內(nèi)雌激素受體α的表達(dá)降低相關(guān),增強的啟動子區(qū)域的DNA甲基化是其可能機(jī)制。
[Abstract]:British scientist David J. P. Baker first proposed the "fetal origins of adult disease" in 1995 (Fetal Origins of Adult Disease, FOAD) hypothesis, then gradually evolved into today's "developmental origins of health and disease" (Developmental Origins of Health and Disease, DOHaD) hypothesis. The hypothesis that humans in the early developmental process in (including fetal, infant, child) experienced adverse factors, tissues and organs will be permanent or procedural changes in structure and function, will affect adult diabetes, metabolic syndrome, cardiovascular disease, occurrence and development of mental and behavior disorders and other chronic non communicable diseases. Endocrine disruptors, as ubiquitous environmental contaminants the development period, relationship between exposure and developmental toxicity of organ system and adult disease susceptibility has attracted more and more attention. Bisphenol A (BPA), is. Before the world manufacturing and one of the most used environmental endocrine disruptors. As a monomer molecule, BPA is mainly used in manufacturing polycarbonate and epoxy resin can be used for polymer materials.BPA food and beverage packaging exposure via food packaging and beverage containers into food and oral intake, such as skin contact and breathing intake is an important exposure pathway. Moreover, studies show that BPA can through the placenta and breast milk transfer to the fetus and newborn, formed a serious threat to human health. Animal studies and human epidemiological research show that developmental exposure to BPA can change the adult neural behavior including learning / memory and emotional adjustment, but the underlying mechanism is still not clear. Previous studies also indicate that developmental BPA exposure can increase the susceptibility of adult diabetes, and also the mechanism is not clear. Study from the pancreas development perspective to explore the possible mechanism of growing rats exposed to BPA induced disorders of glucose metabolism in adulthood, and the potential epigenetic mechanism. This research then explores the developmental effects of exposure to BPA on adult rat learning and memory and anxiety, as well as the influence of the corresponding brain regions the behavior of related gene expression, and to explore the potential epigenetic mechanism. The first part of the epigenetic mediated BPA interference during the development of exposure of rat pancreatic development and induction of adult glucose metabolism disorder Objective: To explore the developmental period of exposure to low dose of BPA influence on the early development of P cells in the pancreas, and the expression of the key gene control pancreatic related development. On this basis, to explore the early exposure of BPA damaged pancreatic development potential epigenetic mechanisms and effects on glucose metabolism in adulthood. Methods: by According to the weight of the pregnant Wistar rats, were randomly divided into control group and BPA exposure group. From the beginning of the first day of pregnancy, daily oral administration of exposed group of pregnant rats 10 g/kg weight of BPA, the control group was given the corresponding solvent control of corn oil. Postnatal rats, expression by immunohistochemistry and quantitative PCR detection pancreatic beta cell development and key regulatory genes; on this basis, from embryonic day 15.5 to observe the expression of key genes in pancreatic pancreatic development; rats after birth to adulthood, tolerance test and insulin tolerance test, glucose utilization, sequential observation in glucose metabolism; from embryonic to adult, using chromatin immunoprecipitation and mass spectrometry, continuous determination of changes in histone modifications and DNA methylation of key regulatory genes in the process of pancreatic development. Results: the embryos were exposed to 10 g/kg/d BPA can reduce the rat pancreatic P cell mass at birth (Beta-cell mass, BCM), and reduce pancreatic development key regulatory genes of pancreatic and duodenal homeobox 1 (pancreatic duodenal, homeobox-1, Pdx1) expression. Further studies showed that the expression of BPA on pancreatic Pdxl effect in the 15.5 day of pregnancy which have already appeared, the pancreas showed PDX1 positive cell fraction decreased.BPA exposure in the embryonic period significantly altered histone Pdxl promoter modification of sub regions. The expression of inactivation; decreasing of histone H3 and H4 acetylation of histone H3 at lysine fourth trimethylation decreased and 9 lysine bimethylation increased; and the inactivation of regulatory trend with the growth and development of fetus were deepened until adulthood however, in this study; Pdxl gene promoter DNA methylation detection had no obvious change; maternal BPA exposure the adult offspring glucose metabolism. Conclusion: developmental exposure to BPA can significantly change the pancreas Gland development key regulatory gene Pdxl promoter histone modification sub region, the transition to the inactive state, thus affecting the early pancreatic P cell development, and ultimately cause glucose metabolic disorder in adulthood. The second part of the BPA development period of exposure to DNA methylation damage in rats learning and memory ability and hippocampal estrogen receptor alpha discussion: developmental exposure to bisphenol A (BPA) effect in offspring rats learning and memory behavior and emotion regulation and exploratory behavior, and to explore the epigenetic mechanism. Methods: Sprague-Dawley pregnancy (SD) rats, were randomly divided into control group and BPA exposure group. After birth to 21 from the beginning of the first day of weaning pregnancy day by intragastric administration exposed rats with 40 g/kg body weight BPA, the control group was given the corresponding solvent control of corn oil. 60 days after the birth of the offspring of rats, rats were determined by Morris water maze learning The learning and memory ability of rat offspring; 85 days after birth, the determination of anxiety in rats and explore the behavior with the elevated plus maze; the concentration of corticosterone in rat serum was measured by ELISA method, the expression level at the same time the application of quantitative PCR method for the determination of offspring in hippocampus of rats in estrogen receptor alpha and estrogen receptor beta; final determination estrogen receptor alpha in rat offspring hippocampus in the promoter region methylation status of BPA exposed offspring rats learning and memory behavior development of epigenetic mechanism. Results: the water maze experiment, developmental exposure to BPA significantly prolonged the first male and female offspring rats in training to find the latent time in exploration platform, then training reduced offspring rats appear in the target quadrant time. The elevated plus maze, developmental exposure to BPA did not produce a system in the offspring of rats No statistically significant changes in behavior; however, BPA exposed offspring corticosterone concentrations in serum of rats compared with the control group, the offspring rats were significantly increased, and this kind of change presents the gender specificity. Compared with the control group, the development period of exposure to BPA significantly reduced the expression level of offspring rat hippocampal estrogen receptor alpha however, estrogen receptor beta did not change significantly. During the development of exposure to BPA also significantly increased the offspring rat hippocampal estrogen receptor alpha gene promoter DNA methylation. Conclusion: developmental exposure to BPA offspring rats learning and memory impaired ability to reduce this kind of damage related with estrogen receptor alpha progeny the expression in the rat hippocampus, enhanced promoter DNA methylation is the possible mechanism.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R114

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