表观遗传介导发育期BPA暴露致大鼠胰腺发育和学习记忆受损的研究

发布时间：2018-01-28 21:40

本文关键词： 双酚A β细胞胰十二指肠同源盒1 组蛋白修饰 DNA甲基化血糖稳态双酚A 学习记忆焦虑行为皮质酮雌激素受体 DNA甲基化　出处：《华中科技大学》2016年博士论文　论文类型：学位论文

【摘要】：英国科学家David J. P. Baker在1995年首次提出了“成人疾病的胎儿起源”(Fetal Origins of Adult Disease,FOAD)假说,后来逐渐演变成了如今的“健康和疾病的发育起源”(Developmental Origins of Health and Disease, DOHaD)假说。该假说认为人类在早期发育过程中(包括胎儿、婴儿、儿童时期)经历不利因素,组织器官在结构和功能上会发生永久性或程序性改变,将会影响成年期糖尿病、代谢综合征、心血管疾病、精神行为异常等慢性非传染性疾病的发生发展。内分泌干扰物,作为广泛存在的环境污染物,其发育期暴露对器官系统的发育毒性及与成年期疾病易感性的关系已引起越来越多的关注。双酚A (BPA),是目前世界范围内制造和使用量最多的环境内分泌干扰物之一。作为单体分子,BPA主要用于生产制造聚碳酸酯和环氧树脂等可用于食品和饮料包装的高分子材料。BPA暴露主要经由食品包装和饮料容器中渗入到食物中从而经口摄入,其他如皮肤接触和呼吸摄入等也是重要暴露途径。而且,研究表明BPA可以经过胎盘和母乳传递给胎儿和新生儿,对人类健康形成严重威胁。动物研究和人群流行病学研究都表明发育期暴露于BPA可改变成年期神经行为包括学习／记忆和情绪调节,但是其潜在的机制尚不明确。前期研究同样表明发育期BPA暴露可增加成年期糖尿病易感性,且机制同样尚不明确。本研究首先从胰腺发育的角度探讨大鼠发育期暴露于BPA致成年期糖代谢紊乱的可能途径,及其中潜在的表观遗传机制。本研究接着探讨发育期暴露于BPA对成年期大鼠学习记忆和焦虑行为的影响,以及对控制相应行为的大脑区域内相关基因表达的影响,并探讨潜在的表观遗传机制。第一部分表观遗传介导BPA发育期暴露干扰大鼠胰腺发育并诱导成年期糖代谢紊乱目的：探讨发育期暴露于低剂量BPA对胰腺内p细胞早期发育的影响,及与此有关的控制胰腺发育的关键基因的表达。在此基础上,探讨BPA暴露引起的早期胰腺发育受损潜在的表观遗传机制及对成年期糖代谢的影响。方法：将受孕的Wistar大鼠按体重匹配后随机分成对照组和BPA暴露组。从妊娠第一天开始,每天经口给予暴露组孕鼠10μg/kg体重的BPA,对照组给予相应的溶剂对照玉米油。大鼠出生后,利用免疫组化和定量PCR检测胰腺β细胞发育及关键调控基因的表达；在此基础上,分离胚胎15.5天胰腺观察胰腺发育关键基因的表达；大鼠出生后至成年,利用葡萄糖耐量试验和胰岛素耐量试验,顺序观察糖代谢情况；从胚胎期至成年,利用染色质免疫共沉淀和质谱法,持续测定胰腺发育过程中关键调控基因的组蛋白修饰和DNA甲基化改变。结果：胚胎期暴露于10μg/kg/d BPA可降低出生时仔鼠胰腺p细胞质量(Beta-cell mass, BCM),并降低胰腺发育关键调控基因胰十二指肠同源盒1(pancreatic duodenal homeobox-1, Pdx1)的表达。进一步研究发现,BPA对胰腺Pdxl表达的影响在妊娠15.5天既己出现,表现为胰腺内PDX1阳性细胞分数降低。BPA暴露在胚胎期显著改变Pdxl启动子区域的组蛋白修饰,使之表达失活；主要表现为组蛋白H3和H4乙酰化降低,组蛋白H3第4位赖氨酸三甲基化降低和9位赖氨酸双甲基化升高；且此种失活调节趋势可随着胎儿生长发育进行性加深,直至成年期；然而,本研究中Pdxl基因启动子区域DNA甲基化检测未见明显改变；母体BPA暴露最终使成年期子代发生糖代谢紊乱。结论：发育期暴露于BPA可显著改变胰腺发育关键调控基因Pdxl启动子区域的组蛋白修饰,使之向失活状态转变,从而影响早期胰腺p细胞发育,最终在成年期引起糖代谢紊乱。第二部分BPA发育期暴露损害大鼠学习记忆能力和海马区雌激素受体α的DNA甲基化目的：探讨发育期暴露于双酚A(BPA)对子代大鼠学习记忆行为和情绪调节及探索行为的影响,并探讨其中的表观遗传学机制。方法：将受孕的Sprague-Dawley (SD)大鼠按体重匹配后随机分成对照组和BPA暴露组。从妊娠第一天开始至出生后21天断乳,每天灌胃给予暴露组大鼠40μg/kg体重的BPA,对照组给予相应的溶剂对照玉米油。子代大鼠出生后60天,利用Morris水迷宫测定大鼠的学习记忆能力；子代大鼠出生后85天,利用高架十字迷宫测定大鼠的焦虑和探索行为；应用ELISA方法测定大鼠血清中的皮质酮浓度,同时应用定量PCR法测定子代大鼠海马组织中雌激素受体α和雌激素受体β的表达水平；最后测定子代大鼠海马中雌激素受体α的基因启动子区域甲基化状态,探讨BPA暴露影响子代大鼠学习记忆行为发育的表观遗传学机制。结果：水迷宫实验中,发育期暴露于BPA首先显著延长了雌雄子代大鼠在获得性训练中找到潜伏平台的时间,其次在探查训练中降低了子代大鼠出现在目标象限的时间。高架十字迷宫实验中,发育期暴露于BPA没有在子代大鼠中产生有统计学意义的行为改变；但是,BPA暴露子代大鼠血清中的皮质酮浓度相比于对照组子代大鼠显著升高,且此种改变呈现出性别特异性。相对于对照组,发育期暴露于BPA显著降低了子代大鼠海马雌激素受体α的表达水平,而雌激素受体β没有明显改变。发育期暴露于BPA还显著升高了子代大鼠海马雌激素受体α基因启动子区域的DNA甲基化。结论：发育期暴露于BPA使子代大鼠学习记忆能力受损,此种受损与子代大鼠海马内雌激素受体α的表达降低相关,增强的启动子区域的DNA甲基化是其可能机制。
[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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R114

【相似文献】