胚胎期双酚A暴露致雄性仔鼠学习记忆降低的分子机制研究

发布时间：2018-06-06 22:57

本文选题：双酚A + 自发性行为　；参考：《山西农业大学》2014年博士论文

【摘要】：[目的]双酚A (BisphenolA, BPA)作为具有雌激素活性的环境内分泌干扰物,是环氧树脂和碳酸乙酯的主要材料,对发育中的中枢神经系统,特别是学习记忆有副作用,但BPA影响学习记忆的机制还不是很清楚。本文主要从表观遗传学、信号通路和突触可塑性三个方面探讨了胚胎期BPA暴露对子代雄性大鼠学习记忆能力影响的分子机制,为进一步阐明BPA的神经毒性机制奠定理论基础,为今后实施科学诊断和靶标药物设计提供实验室依据。[方法]怀孕的Sprague-Dawley大鼠从怀孕第9天(E9)到怀孕第20天(E 20)口服0.05、0.5、5或50 mg/kg-body weight (BW) per day的BPA,选取出生后21天(PND21)的雄性小鼠进行相关试验。采用开场试验、目标识别试验和八臂迷宫试验检测雄性仔鼠的自发性行为和学习记忆能力；采用HE染色、透射电镜、Real-time PCR, Western blotting等技术手段,检测了雄性仔鼠的脑组织形态结构、海马CA1区超微结构、表观遗传相关酶类(DNMTs和HDACs)、ERK信号通路相关分子、突触标记物(synaptophysin, postsynaptic density-95 (PSD-95), spinophilin)、谷氨酸受体(G1uR1和NMDARl) mRNA和蛋白的表达,从而探讨胚胎期BPA暴露引起神经毒性的分子机制。[结果](1)胚胎期BPA暴露影响了出生后雄性仔鼠的生理生化指标,包括增加了出生后第9天、14天和21天雄性小鼠的体重；降低了脑、海马和睾丸的脏体比；增加了血清睾酮浓度,降低了血清雌激素水平；同时,诱导了雄性仔鼠海马组织形态学的病理损伤：海马各区锥体细胞和颗粒细胞明显体积变小,数量减少,细胞间隙增大,神经元皱缩,细胞边缘不清晰。(2)胚胎期BPA暴露影响了出生后雄性仔鼠在开场试验中的自发性行为,包括降低了运动活性、减少了探索行为和理毛行为；损害了目标识别实验中的短期记忆和长期记忆；增加了八臂迷宫中的参考记忆特别是工作记忆错误次数。(3)胚胎期BPA暴露影响了出生后雄性仔鼠海马中相关表观遗传酶的表达,Real-time PCR结果显示BPA暴露抑制了DNA甲基转移酶DNMT1 mRNA和组蛋白去乙酰化酶HDAC1 mRNA表达水平,上调了组蛋白去乙酰化酶HDAC2 mRNA表达水平。(4)胚胎期BPA暴露影响了出生后雄性仔鼠海马中ERK通路相关分子的表达,Western Blotting数据显示,与对照组相比,BPA暴露组雄性小鼠海马中Akt、phospho-Akt、 p44/42 MAPK、phospho-p44/42 MAPK、phospho-CREB、BDNF蛋白表达水平显著降低。(5)胚胎期BPA暴露影响了出生后雄性仔鼠海马CA1区突触超微结构：包括突触间隙变宽、PSD区变薄、突触表面模糊不清、突触囊泡消失；Real-time PCR和Western Blotting实验数据表明,BPA暴露下调了突触标记性分子(synaptophysin, PSD-95,和spinophilin) mRNA和蛋白表达；抑制了谷氨酸受体(G1uR1和NMDARl) mRNA和蛋白表达。[结论]本课题实验结果揭示了胚胎期BPA暴露诱导了出生后雄性仔鼠的学习记忆能力的降低,其分子机制涉及到表观遗传学、ERK通路和突触可塑性的改变,提示BPA影响中枢神经系统和行为发育机制的复杂性。
[Abstract]:[Objective] bisphenol A (BisphenolA, BPA), as an environmental endocrine disruptor with estrogen activity, is the main material of epoxy and ethyl carbonate. It has side effects on the developing central nervous system, especially learning and memory, but the mechanism that BPA affects learning and memory is not clear. This article mainly from epigenetics, signal pathway The three aspects of synaptic plasticity discussed the molecular mechanism of the effects of BPA exposure on the learning and memory ability of the offspring of the offspring of the offspring, which lay a theoretical basis for further clarifying the neurotoxic mechanism of BPA, and provided a laboratory basis for the future implementation of scientific diagnosis and target drug design. [methods] the pregnant Sprague-Dawley rats were ninth from pregnancy. Days (E9) to twentieth days of pregnancy (E 20) oral 0.05,0.5,5 or BPA of 50 mg/kg-body weight (BW) per day, selected male mice of 21 days after birth (PND21). The spontaneous behavior and learning and memory ability of male offspring were detected by open test, target identification test and eight arm labyrinth test; HE staining, transmission electron microscopy, and Rea were used. L-time PCR and Western blotting were used to detect the morphological structure of the brain tissue of the male offspring, the ultrastructure of the hippocampus CA1 region, the epigenetic related enzymes (DNMTs and HDACs), the related molecules of the ERK signaling pathway, the synaptic markers (synaptophysin, postsynaptic density-95, PSD-95), and the glutamate receptors. The expression of protein and protein to explore the molecular mechanism of neurotoxicity induced by BPA exposure during embryonic period. [results] (1) BPA exposure in embryonic stage affects the physiological and biochemical indexes of postnatal male offspring, including increasing the weight of male mice at ninth days, 14 days and 21 days after birth, and reducing the ratio of the visceral body in the brain, hippocampus and testis, and the increase of serum testosterone. At the same time, the serum estrogen level was reduced, and the pathological damage of the hippocampal morphology was induced in male offspring: the pyramidal cells and granulosa cells in the hippocampus were obviously smaller, the number of cells decreased, the intercellular space increased, the neuron crinkled and the cell edge was not clear. (2) the BPA exposure in the embryonic stage affected the male offspring in the opening test after birth. Spontaneous behavior in the test, including reduced activity activity, reduced exploratory behavior and hair handling, impaired short-term and long-term memory in target recognition experiments, increased the number of reference memories in the eight arm maze, especially working memory errors. (3) BPA exposure in the embryonic stage affected the related tables in the hippocampus of postnatal male offspring. The expression of Real-time PCR showed that BPA exposure inhibited the expression level of DNA methyltransferase DNMT1 mRNA and histone deacetylase HDAC1 mRNA, up up the expression level of the histone deacetylase HDAC2 mRNA. (4) BPA exposure at embryo stage affected the expression of ERK pathway related molecules in the hippocampus of postnatal male offspring. Lotting data showed that compared with the control group, the expression level of Akt, phospho-Akt, p44/42 MAPK, phospho-p44/42 MAPK, phospho-CREB, BDNF protein in the hippocampus of the male mice of the BPA exposure group decreased significantly. (5) the BPA exposure in the embryonic stage affected the ultrastructure of the synapse in the hippocampal CA1 area of the male offspring of the postnatal male offspring, including the widening of the synaptic gap, the thinning of the synapse and the synapse. The surface was blurred and synaptic vesicles disappeared; Real-time PCR and Western Blotting experimental data showed that BPA exposure lowered the mRNA and protein expression of the synaptic markers (synaptophysin, PSD-95, and spinophilin), and inhibited the glutamate receptor (G1uR1 and NMDARl) mRNA and protein expression. [Conclusion] the experimental results of this subject revealed the embryo period. BPA exposure induces a decrease in the learning and memory ability of postnatal male offspring, and its molecular mechanism involves epigenetics, ERK pathway and synaptic plasticity, suggesting that BPA affects the complexity of the central nervous system and the mechanism of behavioral development.
【学位授予单位】：山西农业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114

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