食品源铅暴露对发育期海马神经元发育的影响及机理研究

发布时间：2018-11-07 10:30

【摘要】：食品安全与人类健康息息相关,而随着环境问题的突出,食品源性铅暴露屡见不鲜。铅是一种传统的重金属污染物,对神经系统尤其是中枢神经系统具有毒性效应。本研究组的前期工作结果表明,发育期铅暴露会对大鼠大脑认知功能造成一定的损伤,但是其内在具体机制尚未完全明确。近来,有研究表明铅暴露对神经元发育过程有抑制作用,但对其内在调节机制研究甚少。目的:研究铅暴露对Sprague-Dawley(SD)大鼠海马区神经元增殖、分化的影响及相关信号通路;研究铅暴露调节神经元成熟的详细生物分子机制进而推测其致神经退行性疾病的潜在机制。方法:1.SD大鼠按照雌雄1:3配对后随机分为正常对照组和铅暴露组。正常对照组饮用蒸馏水,铅暴露组饮用铅水(250ppm醋酸铅溶于蒸馏水,30mL/d)。实验鼠自胚胎期开始接受铅暴露,出生后至断奶前(出生后21天)通过母乳间接接受铅暴露,断奶后通过饮用铅水直接接受铅暴露。对照组和铅暴露组幼鼠均于出生后第19天至21天通过腹腔注射给予5-溴脱氧尿嘧啶核苷(BrdU),40mg/kg,每天一次,于第22天断头取材。免疫组织化学观察海马区新生神经元数量,Western blot检测经典Wnt通路相关蛋白表达量,Real-time Quantitative PCR(q-PCR)检测经典Wnt通路中mRNA的表达。2.以离体培养的海马神经元为实验材料,从RNA调控水平深入探究铅暴露抑制神经元发育的分子机制。取新生大鼠(出生后24小时内,P0)原代海马神经元进行细胞培养,体外培养第3天开始进行醋酸铅(5μM)处理,连续暴露11天,于第14天收样。Western blot实验和q-PCR实验分别从蛋白水平和mRNA层面检测Kmt2a(MLL1)的表达,同时q-PCR实验检测具有招募功能的长链非编码RNA(LncRNA)的表达。结果:1.铅暴露显著降低SD大鼠海马区新生神经元数目(BrdU标记)。2.铅暴露显著升高磷酸化β-catenin蛋白水平,降低磷酸化GSK-3β蛋白水平,而对GSK-3β和β-catenin总体蛋白水平没有产生显著影响。此外GSK-3β在mRNA水平上显著上升。3.铅暴露显著抑制MLL1蛋白水平和mRNA水平的表达。4.铅暴露可影响H3K4me3在染色质中分布,并且这种改变可能是通过影响具有招募功能的LncRNA表达实现的。结论:本研究从经典Wnt信号通路和靶分子H3K4me3两个分子机制作为切入点共同探究发育期铅暴露对大脑新生神经元的增殖、分化以及成熟两个方面过程的影响。1.发育期铅暴露可能通过抑制经典Wnt通路来影响发育期大鼠海马区新生神经元增殖、分化。2.对神经元发育成熟过程有重要调节作用的MLL1分子的表达受铅暴露影响,同时改变了H3K4me3在染色质上的分布,并且这一过程可能是通过调控具有招募功能的LncRNA实现的。该研究成果从神经元发育的角度为今后关于发育期铅暴露的神经毒性的研究提供了新的视野。
[Abstract]:Food safety is closely related to human health, but with the prominent environmental problems, food-derived lead exposure is common. Lead is a traditional heavy metal pollutant, which has toxic effects on nervous system, especially central nervous system. The preliminary results of this study showed that the brain cognitive function of rats was damaged by lead exposure during developmental period, but its specific mechanism was not completely clear. Recent studies have shown that lead exposure inhibits the development of neurons, but little research has been done on its intrinsic regulatory mechanisms. Objective: to study the effects of lead exposure on the proliferation and differentiation of hippocampal neurons in Sprague-Dawley (SD) rats and the related signal pathways. To study the detailed biomolecular mechanism of lead exposure regulating neuronal maturation and to speculate on the underlying mechanism of neurodegenerative diseases induced by lead exposure. Methods: 1.SD rats were randomly divided into normal control group and lead exposure group. The normal control group drank distilled water, and the lead exposed group drank lead water (250ppm lead acetate dissolved in distilled water, 30mL/d). The mice were exposed to lead from embryonic stage, from birth to before weaning (21 days after birth) through breast milk. After weaning, they were exposed to lead directly by drinking lead water. The young rats in the control group and lead exposure group received intraperitoneal injection of 5-bromodeoxyuridine (BrdU),) 40 mg / kg from 19 to 21 days after birth. Immunohistochemical method was used to detect the number of new neurons in hippocampal area., Western blot was used to detect the expression of classical Wnt pathway associated protein, and Real-time Quantitative PCR (q-PCR) was used to detect the expression of mRNA in classical Wnt pathway. 2. Hippocampal neurons cultured in vitro were used as experimental materials to explore the molecular mechanism of lead exposure inhibiting the development of neurons from the level of RNA regulation. The primary hippocampal neurons of neonatal rats (within 24 hours after birth) were cultured and treated with lead acetate (5 渭 M) on the third day of culture. On the 14th day, the expression of Kmt2a (MLL1) was detected by. Western blot assay and q-PCR assay at the protein level and mRNA level, respectively. Meanwhile, the q-PCR assay was used to detect the expression of long chain non-coding RNA (LncRNA) with recruitment function. The result is 1: 1. Lead exposure significantly decreased the number of neonate neurons (BrdU labeled) in the hippocampus of SD rats. 2. Lead exposure significantly increased the level of phosphorylated 尾-catenin protein, decreased the level of phosphorylated GSK-3 尾-protein, but had no significant effect on the total protein levels of GSK-3 尾 and 尾-catenin. In addition, GSK-3 尾 increased significantly at mRNA level. Lead exposure significantly inhibited the expression of MLL1 protein and mRNA. 4. Lead exposure may affect the distribution of H3K4me3 in chromatin, and this change may be achieved by influencing the expression of LncRNA with recruitment function. Conclusion: in this study, the effects of lead exposure on the proliferation, differentiation and maturation of neonate neurons in the brain were explored from the classical Wnt signaling pathway and the target molecule H3K4me3 as a starting point. 1. Lead exposure during development may affect the proliferation and differentiation of neonatal neurons in the hippocampus of developing rats by inhibiting the classical Wnt pathway. 2. The expression of MLL1 molecules which play an important role in the process of neuronal development and maturation is affected by lead exposure and the distribution of H3K4me3 in chromatin is changed. This process may be achieved by regulating the recruitment of LncRNA. The results provide a new perspective for the study of neurotoxicity of lead exposure in the future from the point of view of neuronal development.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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