围生期双酚A暴露对子代小鼠海马突触结构发育的影响
发布时间：2018-09-18 11:02
【摘要】：环境雌激素是环境中干扰体内激素生成、释放、输送、代谢、结合或消除过程的外源性化合物,表现出拟雌激素或抗雌激素作用。双酚A(Bisphenol A, BPA)是一种典型的环境雌激素,广泛用于、食品和饮料包装材料、金属罐头树脂内膜、牙齿固封剂、添加剂等。BPA能够通过食品和饮料进入人体,也可以从垃圾渗析到周围生态系统污染环境而危害健康。BPA具类雌激素活性,通过与雌激素受体结合干扰内源性雌激素活性。 近来有研究发现,低于风险评估值的BPA对啮齿类动物中枢神经系统发育存在低剂量效应。围生期母体BPA暴露可影响子代大鼠大脑皮层结构以及皮层和海马神经元的形态发育,改变其多种神经行为及其性别分化,说明发育中的脑是对BPA敏感的靶器官。本实验室前期研究发现,围生期母体BPA (0.05～50mg/kg/d)暴露后,仔鼠的活动性、探究、焦虑和抑郁、学习记忆等多种非生殖行为发生改变；体外培养的海马神经元暴露于BPA后快速促进树突丝状的运动性和密度。鉴于突触是脑可塑性变化和信息传递的关键部位,与学习记忆等行为密切相关,而突触结构修饰包括突触界面结构、突触数密度、突触传递等容易受环境和药物的影响,因此,本研究通过围生期母体BPA暴露,观察BPA对雄性仔鼠生后发育过程中海马突触结构的影响,并通过检测相关脑区突触前标志蛋白synapsinI和突触后标志蛋白PSD-95,以及与学习记忆密切相关的突触后谷氨酸NMDA和AMPA受体的表达水平进一步探讨BPA影响突触结构的分子机制。 方法：取清洁级ICR雄鼠(30-35g)和雌鼠(25-30g)适应1周后合笼,母鼠从妊娠第7天至断乳期(产后21天)进行双酚A (Bisphenol A, BPA,0.04、0.4、4mg/kg/day)灌胃染毒,同时设对照组给予溶媒花生油。子代雄鼠每组10只,分别在生后14天、21天、56天处死,按要求取海马组织样品,分别制备电镜超薄切片观察海马CA1区锥体细胞的突触数密度和突触界面参数,应用western-blot和免疫组织化学技术分别检测海马synapsin I、NMDA受体亚基NR1和AMPA受体亚基GluR1蛋白表达。 用Quantity one软件测量目的蛋白条带光密度值,用Image-Pro Plus6.0分析测定突触形态和免疫组织化学图片。实验数据采用SPSS15.0统计软件中的One-way ANOVA法分析,测定值用平均值±标准误差(M±SE)表示,进行t检验,P0.05作为具有统计学差异显著性。 结果：1、围生期母体BPA暴露后,与对照组相比,BPA(0.04和4mg/kg/d)显著减少PND14和PND56的突触数密度(P0.01或P0.001),但对PND21没有显著差异。说明围生期BPA暴露抑制子代小鼠突触形成。 2、围生期母体BPA暴露改变突触界面结构。BPA所有剂量组均极显著增大不同发育阶段的突触间隙宽度(P0.01)；所有剂量组使PSD厚度极显著减小(P0.01)；缩短不同发育阶段的突触活性带长度,其中,生后21天影响最显著(P0.05或P0.01)；但对突触界面曲率影响较小,主要是发育早期(PND14和21)有显著上升(P0.05)。说明突触界面参数对BPA的敏感程度不一样,其中突触间隙和PSD厚度最为敏感。 3、Western blot分析结果发现,围生期BPA暴露,所有剂量组显著降低生后21天和56天synapsin I蛋白表达(P0.05或P0.01)；显著下调PSD-95蛋白表达,其中生后56天下降最显著(P0.05,P0.01或P0.001)；BPA(0.4和4mg/kg/d)显著降低PND21的NR1表达,所有剂量组使56天表达极显著下降(P0.05,P0.01或P0.001),但对PND14无显著影响；GluRl蛋白表达被抑制,低剂量组和高剂量组显著或极显著降低发育阶段的表达量(P0.05或P0.01)。 4、免疫组化技术分析结果与WB结果相似。 结论：低于日允许摄入剂量(tolerable daily intake,0.05mg/kg/d)的BPA也可影响脑的突触发育和突触可塑性；围生期母体BPA暴露影响仔鼠发育期海马突触结构,并降低兴奋性氨基酸NMDA和AMPA受体、突触蛋白PSD-95和synapsin I蛋白的表达。推测这可能是围生期母体BPA暴露影响仔鼠发育期和成年后多种行为的原因之一。
[Abstract]:Bisphenol A (BPA) is a typical environmental estrogen widely used in food and beverage packaging materials, canned metal resin lining, dental sealants, etc. BPA can enter the human body through food and beverage, and can also be leached from landfill to the surrounding ecosystem to pollute the environment and endanger health. BPA has estrogenic activity and interferes with endogenous estrogenic activity by binding with estrogen receptors.
Recent studies have found that BPA below risk assessment has a low-dose effect on the development of the central nervous system in rodents. Perinatal exposure to BPA can affect the cortical structure and morphological development of cortical and hippocampal neurons in offspring rats, alter a variety of neurobehavioral and sexual differentiation, suggesting that the developing brain is BPA. Sensitive target organs. Previous studies in our laboratory found that after exposure to maternal BPA (0.05-50mg/kg/d) during perinatal period, the activity, exploration, anxiety and depression, learning and memory and other non-reproductive behaviors of offspring were altered; in vitro cultured hippocampal neurons exposed to BPA rapidly promoted dendritic filamentous mobility and density. The changes of plasticity and the key parts of information transmission are closely related to learning and memory. Synaptic modifications include the structure of synaptic interface, number density of synapses, synaptic transmission and so on, which are susceptible to the influence of environment and drugs. Therefore, this study observed the effects of BPA on the hippocampal synapses of male offspring during postnatal development through exposure of maternal BPA. The effects of BPA on synaptic structure were further explored by detecting synapsin I and PSD-95, as well as the expression levels of NMDA and AMPA receptors, which are closely related to learning and memory.
METHODS: Clean grade ICR male rats (30-35g) and female rats (25-30g) were caged after one week. Bisphenol A (BPA, 0.04, 0.4, 4mg/kg/day) was administered to female rats from the 7th day of pregnancy to the 21st day of postpartum. The control group was given solvent peanut oil. Ten male offspring in each group were sacrificed on the 14th, 21st and 56th days after birth. The synaptic density and synaptic interface parameters of hippocampal CA1 pyramidal cells were observed by ultrathin sections. The expression of synapsin I, NMDA receptor subunit NR1 and AMPA receptor subunit GluR1 in hippocampal CA1 pyramidal cells was detected by Western blot and immunohistochemistry.
Quantity one software was used to measure the optical density of the target protein bands, and image-Pro Plus 6.0 was used to analyze the synaptic morphology and immunohistochemical images. Sex.
Results: 1. Compared with the control group, BPA (0.04 and 4 mg/kg/d) significantly decreased the synaptic number density of PND14 and PND56 (P 0.01 or P 0.001), but there was no significant difference in PND21 between the two groups.
2. Perinatal maternal exposure to BPA altered the structure of synaptic interface. All BPA doses significantly increased the width of synaptic gap at different developmental stages (P 0.01); all doses significantly decreased the thickness of PSD (P 0.01); shortened the length of synaptic active bands at different developmental stages, of which 21 days after birth had the most significant effect (P 0.05 or P 0.01); but on the process. Contact curvature had little effect on BPA, mainly due to a significant increase in early development (PND14 and 21) (P 0.05).
3. Western blot analysis showed that BPA exposure significantly decreased the expression of synapsin I protein at 21 and 56 days postnatal (P 0.05 or P 0.01); significantly decreased the expression of PSD-95 protein at 56 days postnatal (P 0.05, P 0.01 or P 0.001); BPA (0.4 and 4 mg/kg/d) significantly decreased the expression of NR1 at 56 days postnatal. The expression of GluRl protein was inhibited, and the expression of GluRl protein in low dose group and high dose group was significantly or extremely significantly decreased at the development stage (P 0.05 or P 0.01).
4, the results of immunohistochemical technique were similar to those of WB.
CONCLUSION: BPA at a dose below tolerable daily intake (0.05 mg/kg/d) can also affect synaptic development and synaptic plasticity in the brain, and maternal BPA exposure during perinatal period affects synaptic structure in the hippocampus of offspring rats and decreases the expression of excitatory amino acid NMDA and AMPA receptors, synaptic protein PSD-95 and synapsin I. It is one of the reasons that perinatal maternal BPA exposure affects the development of offspring and adult behavior.
【学位授予单位】：浙江师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114
本文编号：2247707
[Abstract]:Bisphenol A (BPA) is a typical environmental estrogen widely used in food and beverage packaging materials, canned metal resin lining, dental sealants, etc. BPA can enter the human body through food and beverage, and can also be leached from landfill to the surrounding ecosystem to pollute the environment and endanger health. BPA has estrogenic activity and interferes with endogenous estrogenic activity by binding with estrogen receptors.
Recent studies have found that BPA below risk assessment has a low-dose effect on the development of the central nervous system in rodents. Perinatal exposure to BPA can affect the cortical structure and morphological development of cortical and hippocampal neurons in offspring rats, alter a variety of neurobehavioral and sexual differentiation, suggesting that the developing brain is BPA. Sensitive target organs. Previous studies in our laboratory found that after exposure to maternal BPA (0.05-50mg/kg/d) during perinatal period, the activity, exploration, anxiety and depression, learning and memory and other non-reproductive behaviors of offspring were altered; in vitro cultured hippocampal neurons exposed to BPA rapidly promoted dendritic filamentous mobility and density. The changes of plasticity and the key parts of information transmission are closely related to learning and memory. Synaptic modifications include the structure of synaptic interface, number density of synapses, synaptic transmission and so on, which are susceptible to the influence of environment and drugs. Therefore, this study observed the effects of BPA on the hippocampal synapses of male offspring during postnatal development through exposure of maternal BPA. The effects of BPA on synaptic structure were further explored by detecting synapsin I and PSD-95, as well as the expression levels of NMDA and AMPA receptors, which are closely related to learning and memory.
METHODS: Clean grade ICR male rats (30-35g) and female rats (25-30g) were caged after one week. Bisphenol A (BPA, 0.04, 0.4, 4mg/kg/day) was administered to female rats from the 7th day of pregnancy to the 21st day of postpartum. The control group was given solvent peanut oil. Ten male offspring in each group were sacrificed on the 14th, 21st and 56th days after birth. The synaptic density and synaptic interface parameters of hippocampal CA1 pyramidal cells were observed by ultrathin sections. The expression of synapsin I, NMDA receptor subunit NR1 and AMPA receptor subunit GluR1 in hippocampal CA1 pyramidal cells was detected by Western blot and immunohistochemistry.
Quantity one software was used to measure the optical density of the target protein bands, and image-Pro Plus 6.0 was used to analyze the synaptic morphology and immunohistochemical images. Sex.
Results: 1. Compared with the control group, BPA (0.04 and 4 mg/kg/d) significantly decreased the synaptic number density of PND14 and PND56 (P 0.01 or P 0.001), but there was no significant difference in PND21 between the two groups.
2. Perinatal maternal exposure to BPA altered the structure of synaptic interface. All BPA doses significantly increased the width of synaptic gap at different developmental stages (P 0.01); all doses significantly decreased the thickness of PSD (P 0.01); shortened the length of synaptic active bands at different developmental stages, of which 21 days after birth had the most significant effect (P 0.05 or P 0.01); but on the process. Contact curvature had little effect on BPA, mainly due to a significant increase in early development (PND14 and 21) (P 0.05).
3. Western blot analysis showed that BPA exposure significantly decreased the expression of synapsin I protein at 21 and 56 days postnatal (P 0.05 or P 0.01); significantly decreased the expression of PSD-95 protein at 56 days postnatal (P 0.05, P 0.01 or P 0.001); BPA (0.4 and 4 mg/kg/d) significantly decreased the expression of NR1 at 56 days postnatal. The expression of GluRl protein was inhibited, and the expression of GluRl protein in low dose group and high dose group was significantly or extremely significantly decreased at the development stage (P 0.05 or P 0.01).
4, the results of immunohistochemical technique were similar to those of WB.
CONCLUSION: BPA at a dose below tolerable daily intake (0.05 mg/kg/d) can also affect synaptic development and synaptic plasticity in the brain, and maternal BPA exposure during perinatal period affects synaptic structure in the hippocampus of offspring rats and decreases the expression of excitatory amino acid NMDA and AMPA receptors, synaptic protein PSD-95 and synapsin I. It is one of the reasons that perinatal maternal BPA exposure affects the development of offspring and adult behavior.
【学位授予单位】：浙江师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114
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