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题名: 二恶英对乙酰胆碱酯酶活性的影响及分子机制
作者: 徐海明
学位类别: 博士
答辩日期: 2014-12
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 赵斌 ; 谢群慧
关键词: 二恶英,乙酰胆碱酯酶,芳香烃受体,转录调控,神经元,dioxins ; acetylcholinesterase ; aryl hydrocarbon receptor ; transcriptional regulation ; neuron
其他题名: The Effects of Dioxins on Acetylcholinesterase Enzymatic Activity and the Underlying Molecular Mechanisms in Vitro
学位专业: 环境科学
中文摘要:     研究表明,二恶英类长期暴露导致暴露人群认知功能损伤。但是,其中的机理尚不明确。胆碱能神经传导系统在高级脑功能(如认知、学习、记忆力和注意力等)的维持中发挥着重要作用。有研究表明,大鼠孕期暴露2,3,7,8-四氯二苯并-对-二恶英(2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD)后子鼠小脑内胆碱能神经传导系统关键水解酶-乙酰胆碱酯酶(acetylcholinesterase,AChE)的酶活性显著降低,并推测其原因可能与TCDD 导致的甲状腺功能减退有关。然而,我们尚不清楚二恶英长期暴露对人神经系统中AChE 酶活性是否存在影响。本论文以人源神经母细胞瘤细胞SK-N-SH 为主要实验模型,系统研究了二恶英对该细胞内AChE 酶活性的影响及分子机制。
     实验结果表明,在一定浓度(≥10-10 mol•L-1)和时间范围(≥24 h)内,TCDD暴露显著抑制SK-N-SH 细胞内AChE 的酶活性。而与有机磷农药等AChE 抑制剂不同,TCDD 并不直接作用于AChE 催化活性中心,其对AChE 酶活性的抑制效应有可能是通过转录途径实现的。
    目前普遍认为二恶英类的毒理效应主要是由芳香烃受体(AhR)及其下游的信号通路介导的。在SK-N-SH 细胞内,我们发现AhR 的抑制剂可以逆转TCDD对AChE 酶活性的抑制作用,说明AhR 是上述作用的一个核心信号分子。我们进一步阐明了AhR 下游的信号分子在下调AChE 表达水平中发挥的作用。结果表明,二恶英反应元件(dioxin responsive element,DRE)以及交叉信号通路-cAMP通路中的cAMP 反应元件(CRE)介导的转录调控都参与了TCDD 对AChE 转录表达的抑制作用。具体地说,在DRE 介导的转录抑制方面,我们使用生物信息学、点突变及凝胶阻滞等实验手段,确定了人ACHE 启动子区存在有功能的DRE 序列,并介导了TCDD 对AChE 转录表达的抑制作用。另一方面,因为cAMP信号通路是典型的神经源性AChE 表达调控的主要机制之一,所以我们进一步研究了cAMP 信号通路在TCDD 导致的AChE 转录抑制中发挥的作用。结果表明,TCDD 可以显著抑制SK-N-SH 细胞内CRE 结合蛋白(CREB)的磷酸化水平,进而干扰CREB 对AChE 转录水平的上调作用;同时cAMP 信号通路的激动剂可以逆转 TCDD对AChE 表达水平的抑制作用。由此初步阐明了TCDD通过AhR抑制AChE 转录表达的分子机制。
    综上所述,二恶英可能通过 AhR 及其下游的一系列信号转导机制下调人源神经元内AChE 的表达水平,并进一步抑制AChE 的酶活性。说明二恶英可以直接干扰人胆碱能神经转导过程中的关键酶AChE。这一新发现补充了二恶英对认知功能损伤机制的理解,并提出ACHE 是AhR 信号通路下游的靶基因。
英文摘要:     Research has shown that long terms exposure to dioxins results in certain cognitive functioning deficits in exposed populations. However, the mechanisms remain unclear. Cholinergic neurotransmission system plays an important role in the maintenance of advanced brain function, including cognition, learning, memory, and attention. Previous research showed that maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during the perinatal period significantly suppressed acetylcholinesterase (AChE) enzymatic activity of their offspring, which was a key hydrolase in cholinergic neurotransmission. It suggested that this suppressive effect might be related to the hypothyroid conditions of rat offspring. However, whether long terms exposure to dioxins affects AChE enzymatic activity in the human nervous system is still unclear. In this study, human neuroblastoma SK-N-SH cells were used to investigate the effects and molecular mechanisms of dioxins on AChE enzymatic activity.
      The results showed that TCDD significantly inhibited AChE enzymatic activity in SK-N-SH at concentrations higher than 10-10 mol·L-1 after 24 hours. Unlike organophosphorus pesticides (typical AChE inhibitors), TCDD did not directly act on the catalytic active center of AChE, but probably through transcriptionaldown-regulation.
      Current thinking suggests that the toxic effects of dioxins are primarily mediated
by aryl hydrocarbon receptor (AhR) and downstream signaling pathways. In SK-N-SH cells, AhR inhibitor counteracted TCDD-induced suppressive effect on AChE enzymatic activity, suggesting that AhR was one of the key signal molecules in AChE transcriptional down-regulation. The roles of downstream signaling pathways of AhR on down-regulation of AChE expression were further elucidated. The transcriptional regulations mediated by dioxin responsive element (DRE) and cAMP response element (CRE) in cAMP signaling pathway (had crosstalk with AhR signaling pathway) were involved in the inhibitory effect of TCDD on AChE transcriptional regulation. Specifically, in the aspect of transcriptional down-regulation mediated by DRE, using bioinformatics, site mutation and gel retardation methods, several functional DRE sites in human ACHE promoter sequence were identified. Subsequent experiments suggested that these functional DRE sites mediated the inhibitory effect of TCDD on AChE transcriptional regulation. On the other hand, the possible role of cAMP signaling pathway in AChE transcriptional down-regulation caused by TCDD was further investigated because cAMP pathway was one of the main mechanisms of AChE expression regulation in neurons. The results showed that TCDD exposure significantly decreased the phosphorylation level of CRE-binding protein (CREB), and thus disrupted CREB-induced AChE transcriptional up-regulation in SK-N-SH cells. In addition, the agonist of cAMP signaling pathway counteracted the suppressive effects of TCDD on AChE expression level. These preliminary results help elucidated the molecular mechanisms of the effects of TCDD on AChE transcriptional down-regulation.
      In conclusion, dioxins suppressed the expression level of AChE and further decreased the enzymatic activity of AChE in human neuroblastoma cells via AhR and a series of downstream signaling pathways. These results indicated that dioxins could disrupt AChE enzymatic activity directly. This study enhances the understanding of the mechanisms of cognitive impairments caused by dioxins, and proposes that ACHE is a novel target gene of AhR.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34451
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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Recommended Citation:
徐海明. 二恶英对乙酰胆碱酯酶活性的影响及分子机制[D]. 北京. 中国科学院研究生院. 2014.
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