【作者】 叶银萍；

【导师】 徐晓虹；

【作者基本信息】 浙江师范大学 ， 生态学， 2010， 硕士

【摘要】 双酚A(bisPhenol A, BPA)是一种具有代表性的内分泌干扰物,具有弱雌激素活性,广泛用于食品和饮料的包装材料碳酸聚酯、环氧树脂、牙齿填充剂及相关产品的制造。目前,BPA对中枢神经系统的影响越来越受到关注。研究发现BPA不仅可影响脑形态结构的发育,还可影响脑内神经递质系统以及神经行为的发育。树突发育是脑发育的重要过程,其结构和特性决定突触信息的传递功效。发育早期树突分支的生长和结构变化迅速,参与突触发生、树突分支形成,以及树突棘的发育。树突分支过度生长可能导致神经系统功能异常。内源性雌激素影响树突发育和突触发生,因此推测具有雌激素活性的BPA也可能会影响树突的发育。谷氨酸NMDA受体在脑内广泛分布,并且与中枢神经系统的发育密切相关。有研究表明,NMDA受体参与神经回路的形成和学习记忆过程,可以调节神经元的存活,轴突和树突的结构发育以及突触的可塑性等,为所有兴奋性突触的突触后必需组份。而雌激素影响树突发育与NMDA受体有关。鉴于NMDA受体在树突发育中的重要作用,本研究通过BPA染毒体外培养的海马神经元,观察BPA急性和长时暴露对树突形态发育、NMDA受体亚基表达和磷酸化、以及雌激素受体β(Estrogen RecePtor beta, ERβ)表达的影响,以确定发育期BPA暴露是否改变树突形态发生,并影响神经元NMDA受体和雌激素受体的表达,探讨BPA影响神经元树突形态发育是否与NMDA受体相关,雌激素受体是否参与了这一过程,为探索BPA影响中枢神经系统发育奠定基础。方法：采用腺病毒感染体外培养第5天(5 DIV)的海马神经元,使其表达绿荧光,通过活细胞成像观测BPA染毒对海马神经元树突形态动力学变化。实验分5个组：对照组(DMSO), BPA各剂量组(浓度分别为1、10、100、1000 nM)。BPA急性暴露时间为0.5 h,慢性暴露时间为24 h。染毒后,在荧光显微镜下对神经元(7DIV)进行活细胞成像,每2 min拍摄一张图片,共11张1组,动态显现神经元树突的生长。采用Image-Pro Plus分析图像,测定树突丝的运动性(μm/20 min)、树突丝密度以及树突分支总长度。用10μM雌激素受体阻断剂ICI 182,780预处理海马培养物0.5 h,然后再进行BPA染毒,以分析BPA对树突形态发育的影响时否通过雌激素受体进行。通过BPA和10nME2的共同暴露,分析BPA与雌激素对树突发育的共同作用。为了进一步分析BPA影响树突发育的机制,采用细胞免疫染色法,用兔来源多克隆NR1、NR2B、P-NR2B(PhosPhorylated NR2B)和ERβ抗体检测了NMDA受体NR1、NR2B、磷酸化NR2B亚基以及ERβ蛋白的表达,并经Image-Pro Plus分析图像,测定各组细胞的光密度值。结果：1.BPA(10～1000 nM)急性暴露0.5 h快速增加树突丝的运动性和密度(P<0.05,P<0.01或P<0.001),该作用可以被雌激素受体阻断剂抑制(P<0.001)；但如果BPA与E2(10 nM)共同给药,BPA或E2单独给药诱导的树突运动性和密度的增加被抑制。BPA(10、100 nM)长时暴露24 h显著增加树突丝的运动性和密度以及树突分支总长度(P<0.001),该作用也可被雌激素受体阻断剂抑制(P＜0.05); BPA与E2 (10nM)共同给药,BPA或E2单独给药表现的增强作用被抑制(P<0.05)。2. BPA (10～1000 nM)急性暴露0.5 h,没有改变NR1、NR2B的表达,但却剂量依赖性地上调了磷酸化NMDA受体2B亚基(P-NR2B)的表达(P<0.05或P＜0.001),这种上调作用可被雌激素受体阻断剂抑制(P＜0.01)。E2 (10nM)增加磷酸化NR2B的表达,如果BPA与E2共同给药,BPA或E2单独给药诱导的磷酸化NR2B表达上调被抑制(P<0.001)。BPA(100、1000 nM)暴露24 h显著下调NR1、NR2B亚基的表达(P<0.001),该作用不受雌激素受体阻断剂的影响；E2 (10 nM)上调NR1、NR2B亚基的表达,如果BPA与E2共同给药, E2单独作用诱导NR1、NR2亚基表达的增加受到抑制。3. BPA (10～1000 nM)急性暴露0.5 h没有引起ERβ蛋白表达的改变,而长时暴露24 h可以上调ERβ蛋白的表达(P<0.001),雌激素受体阻断剂可以抑制该作用(P<0.01)；BPA与E2共同给药与BPA或E2单独给药对ERp表达的影响无明显差异,均可诱导ERβ表达上调。结论：BPA急性暴露可以快速增加体外培养海马神经元树突丝的运动性和密度,促进NMDA受体2B亚基的磷酸化,但不改变海马神经元NR1,NR2B亚基和ERβ蛋白的表达。雌激素受体可能参与了BPA这些作用,由于核内雌激素受体介导的效应产生作用较慢,推测BPA快速作用可能通过膜雌激素受体进行。BPA慢性暴露增加树突丝运动性和密度,以及树突分支总长度,促进ERβ蛋白表达,却下调NR1,NR2B亚基的表达。雌激素受体可能参与了BPA增加树突丝运动性和密度、树突分支总长度,以及上调ER表蛋白表达的作用,而BPA对NR1、NR2B表达的影响可能通过其它途径进行。与E2相似,无论急性或慢性暴露,BPA都可增加树突丝运动性和密度以及磷酸化NR2B的表达；而BPA与E2共同暴露时,BPA或E2单独给药对树突丝运动性和密度以及磷酸化NR2B表达的增加作用被抑制,二者表现出相互拮抗作用。综上所述,BPA快速作用影响树突形态发育及NMDA受体的磷酸化活性,该作用可能通过膜相关雌激素受体的介导。慢性暴露不仅可影响树突形态的发育,也可改变NMDA受体蛋白的表达,从而对脑发育产生重要影响。更多还原

【Abstract】 Bisphenol A (BPA) is one of the typical environmental endocrine disruptors, which has a weak estrogen activity. It is widely used to manufacture food and beverage packaging materials such as polycarbonate plastic, epoxy resin, dental prostheses and sealants, and other related products. At present, the effects of BPA on the central nervous system has been taken more and more attention. BPA can not only affect the brain estrogen synthesis and receptor expression, but also the neurotransmitter systems and the morphologyical development of the brain. During the development of brain dendritic development are important processes. Its structure and characteristics determine the efficiency of synaptic trasmission. Motile dendritic filopodial processes are thought to participate in synaptogenesis, dendritic branching and the development of spines. Excessive growth of dendritic branches may lead to nervous system dysfunction. During the early development, dendritic branches grow and stuctural change rapidly. Excessive growth of dendritic branches will be disappeared for the loss of domination of afferent fibers. Endogenous estrogen can affect the dendritic development and synaptogenesis, BPA which has estrogenic activity may affect the dendritic development. N-methy-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor. It is invoved in a wide variety of processes in the central nervous system (CNS) including learning and memory process, neuronal survival, axon and dendrites stuctural development, and synaptogenesis and synaptic plasticity. Studies showed that NMDA receptor involved in the effect of dendritic development induced by estrogen. Because the important role of NMDA in the dendritic development, we use cultured hippocampal neurons to illuminate the effects of BPA. In this study, we dectect the rapid and slow effects of BPA on the dendritic morphology, the expression of NMDAR subunits NR1, NR2B and phosphorylation of NR2B, and the expression of Estrogen Receptor beta (ERβ).Methods:During the morphological detection of hippocampal neurons, we use adenovirus recombinants encoding EGFP (Ad-EGFP) to infect the cultured hippocampal neurons (5 days in virto),24-48 h after the infection EGFP can be detected. We set five groups:control and BPA (1,10,100,1000 nM BPA,0.01%DMSO as a vehicle). After application of BPA, image of live cells were captured every 2 min for 20 min, using an inverted fluorescent microscope. All images were analysised using Image Pro-Plus software. We measured filopodial motility and dendritic filopodia density and the total length of the dendritic. In order to analyze whether estrogen receptor regulation was involved in the rapid effect of BPA, cultures were pretreated by 10μM antagonist of estrogen receptor ICI 182,780 30 min prior the application of BPA. In order to analyze the interaction the BPA and E2, cultures were co-treated by BPA and E2.For further analysis of the mechanism of BPA effect on dendritic development, the detection of protein was carried out on cultured hippocampal neurons (7 DIV). The methods of BPA application and exprimental group were consistent with the above. For immunocytochemical staining, primary antibody NR1, NR2B, p-NR2B (phosphorylated NR2B) and ERβwere used to dectect the expression of protein. Image Pro-Plus software was used to analysis the ptical density of the neurons.Results:1.0.5 h acute exposure to BPA (10～1000 nM) promoted the motility and density of dendritic filopodia (P＜0.05, P＜0.01 or P＜0.001), and this effect can be inhibited by antagonist of ERs (P＜0.001); 17β-E2(10 nM) treatment significantly increased motility and density of dendritic filopodia, when the hippocampal cultures was co-treated by BPA (100 nM) with E2 (10 nM), the increased of filopodial motility induced by 17β-E2 was obviously attenuated (P＜0.01).24 h exposure to BPA,100 nM BPA promoted the motility and density of dendritic filopodia (P＜0.05, P＜0.01 or P＜0.001), and also the total length of the dendritic (P＜0.05), antagonist of ERs completely inhibited the increases in the filopodial motility, the density of filopodia and the total length of the dendritic induced by BPA (P＜0.05); E2 (10 nM) treatment significantly increased motility and density of dendritic filopodia, and also the total length of the dendritic, when the hippocampal cultures was co-treated by BPA (100 nM) with 17β-E2 (10 nM), the increased of filopodial motility and density and total length of the dendritic induced by 17β-E2 was obviously attenuated (P＜0.05).2.0.5 h treatment with BPA (10～1000 nM) did not change the immunoreactive intensity of NRl and NR2B, but significantly increased the immunoreactive intensity of phosphorylated NR2B (p-NR2B; P＜0.05, P＜0.01, P＜0.001, P＜0.001), the increasement was completely eliminated by antagonist of ERs (P＜0.01); while co-treatment of BPA (100 nM) with 17β-E2 (10 nM) markedly suppressed the immunoreactive intensity of phosphorylation of NR2B induced by 17p-E2 (P＜0.01).24 h treatment with BPA (10 to 1000 nM) significantly decreased the expression of NR1 and NR2B (P＜0.001), but antagonist of ERs did not change the depression of NR1 and NR2B induced by BPA; while co-treatment of BPA (100 nM) with 17β-E2 (10 nM) markedly suppressed the immunoreactive intensity of NRl and NR2B induced by 17β-E2.3.0.5 h acute exposure to BPA did not change the immunoreactive intensity of ERβ. But 24 h treatment with BPA (10～1000 nM) significantly increased the expression of ERβ(P＜0.001), antagonist of ERs markedly suppressed the immunoreactive intensity of ERβinduced by 17β-E2 (P＜0.01).17β-E2(10 nM) increased the expression of ERβ, when co-treatment of BPA (100 nM) with 17β-E2(10 nM) increased the immunoreactive intensity of ERp.Conclusions:Acute exposure to BPA promoted the motility and density of dendritic filopodia as well as the phosphorylation of NR2B, but not affect the expressions of NR1, NR2 and ERβ. For the effect of nuclear estrogen receptor needs more time, suggesting that rapid modulation of BPA involvement of the membrane-associated ER.24 h slowly exposure to BPA promoted the motility and density of dendritic filopodia and the total length of dendritic, and changed the expression of NRl, NR2 and ERβ. These results indicated involvement of ER mediation, but BPA affect NRl, NR2B expression may be through other ways.Acute or slowly exposure to BPA, similar to E2, can promoted the motility and density of dendritic filopodia as well as phosphorylation of NR2B. While co-treatment of BPA (100 nM) with 17β-E2 (10 nM), a suppression was observed. While co-treatment of BPA (100 nM) with 17β-E2 (10 nM), the increased of filopodia motility and density and phosphorylation of NR2B was markedly suppressed.In conclusion, BPA can rapidly effect the development of dendritic filopodia, and this rapid modulation may involve the membrane-associated ER. Slowly exposure to BPA not only effect the development of dendritic filopodia, but also change the expression of NMDA receptor, and then exert an important impact on the brain development.更多还原