二恶英皮肤毒理机制及芳香烃受体配体多样性研究
文献类型：学位论文
| 作者 | 胡芹 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 赵斌 |
| 关键词 | 芳香烃受体 二恶英 细胞分化 波形蛋白 人参皂苷 Aryl hydrocarbon receptor dioxin cell differentiation vimentin ginsenosides |
| 其他题名 | The study of skin toxicology of dioxin and ligand diversity of aryl hydrocarbon receptor |
| 学位专业 | 环境科学 |
| 中文摘要 | 2,3,7,8-四氯代二苯并二恶英（TCDD）及相关的多卤代有机污染物的慢性暴露在现代生活中越来越普遍，是目前在持久性有机污染物中研究最为广泛也是最为深入的物质。TCDD 与芳香烃受体（AHR）具有极强的亲和力，该受体是一种配体依赖的转录因子，可介导多种结构的化合物的很多生物学和毒理学效应，并参与一些重要的生物学过程，如信号转导、细胞分化、细胞凋亡等，是二恶英毒理学研究中最受关注的受体。探讨TCDD 作用的分子靶点及信号通路干扰机制将有助于理解这类化合物的风险性，并提高对这类化合物作用机制的认识。研究表明，氯痤疮是人体接触二恶英后最特异和灵敏的标志性病变。为了研究TCDD导致该病变的分子机制以及生物靶点，我们利用shotgun 蛋白组学分析，发现TCDD 暴露能导致人角质细胞蛋白质表达谱发生巨大变化。该研究中，三种角质 细胞株的蛋白质水平的变化显示细胞分化标志物的显著降低，如微丝聚集蛋白（FLG），角质蛋白1（KRT1）和角质蛋白10（KRT10）。表皮生长因子（EGF）与TCDD 复合刺激加大了这些标志物和另外几种蛋白质的变化。我们还发现，波形蛋白（VIM）是角质细胞中唯一可以被TCDD 诱导却被EGF 逆转的蛋白，并表明VIM 的表达可能与AHR 信号通路相关。尽管TCDD 通常被认为能刺激角质细胞分化，蛋白质组学分析显示TCDD 与细胞分化无关，并提示细胞分化标志物蛋白水平的降低有助于TCDD 诱导细胞过度分层。为了进一步完善对AHR 信号通路以及下游效应机制的理解，本文还研究了一系列效应已知的天然化合物，如人参皂苷等，对AHR 信号通路的影响。通过配体-DNA 结合分析、凝胶阻滞实验、报告基因分析和分子对接等手段，我们证明了部分人参皂苷可以直接结合并激活豚鼠胞质AHR，并且可以激活或抑制重组细胞系中AHR 依赖的荧光素酶报告基因的表达。比较研究表明人参皂苷激活AHR 相关基因表达的能 力在豚鼠、大鼠、小鼠和人的细胞系中具有明显的种属特异性，小鼠细胞系对人参皂苷的响应明显低于大鼠和豚鼠细胞系，但小鼠细胞中的内源性基因细胞色素P4501A1（CYP1A1）仍然能为人参皂苷所诱导。该研究发现，人参皂苷是一类新型的天然来源的AHR 激活剂，该研究结果大大扩展了对AHR 配体结构多样性及其生物功能的认识，结合人参皂苷等化合物的已知功能将有助于我们探索AHR 介导的二恶英类化合物未知的效应及作用机制。 |
| 英文摘要 | Chronic exposure to 2,3,7,8-tetrachlorodibeno-p-dioxin (TCDD) and related polyhalogenated organic pollutants occurs as a consequence of modern life. TCDD is the most potent aryl hydrocarbon receptor (AHR) ligand of its series. The studies about the AHR and its function almost focus on TCDD. The AHR is a ligand-dependent transcription factor that mediates many of biological and toxicological actions of structurally diverse chemicals. Exploring the cellular and molecular basis for their action is anticipated to help understand the risk they pose and the underlying mechanisms. Chlorance is considered to be the most specific and sensitive biomarker of dioxins intoxication. A basis for the striking change in human keratinocyte colony morphology due to TCDD exposure has been investigated by shotgun proteomics. Concentrating on changes in protein levels among three cell strains has revealed significant decreases in the differentiation markers like filaggrin(FLG), keratin 1 (KRT1) and keratin 10 (KRT10). EGF treatment in concert with TCDD enhanced the changes in these markers and several other proteins. The only protein stimulated by TCDD in all three strains and reversed by EGF in them was vimentin (VIM), not previously observed to be in the AHR response domain. Although TCDD is often proposed to enhance keratinocyte differentiation, proteomic analysis reveals it uncouples the differentiation program and suggests that reduced levels of differentiation marker proteins contribute to the observed excessive stratification it induces. In addition, we also examined the ability of a series of naturally occurring compounds including ginsenosides, to bind to and activate/inhibit the AHR and AHR signal transduction. Utilizing a combination of ligand-DNA binding assay, gel retardation assay, reporter gene analysis and molecular docking, we demonstrated the ability of selected ginsenosides to directly bind to and activate the guinea pig cytosolic AHR, and to stimulate/inhibit AHR-dependent luciferase gene expression in a recombinant guinea pig cell line. Comparative studies revealed significant species differences in the ability of ginsenosides to stimulate AHR-dependent gene expression in guinea pig, rat, mouse and human cell lines. Not only did selected ginsenosides preferentially activate the AHR from one species and not others, mouse cell line was also significantly less responsive to these chemicals than rat and guinea pig cell lines, but the endogenous gene CYP1A1 could still be inducted in mouse cell line. Overall, the ability of these compounds to stimulate AHR signal transduction demonstrated that these ginsenosides are a new class of naturally occurring AHR agonists. These results greatly expand the understanding of structural diversity and biological function of AHR ligands. Combined with the known functions of ginsenosides, we can explore more biological roles and the unknown mechanisms of AHR, as well as the interactionship of AHR with other signal transductions. |
| 公开日期 | 2015-06-12 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/13458]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 胡芹. 二恶英皮肤毒理机制及芳香烃受体配体多样性研究[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：生态环境研究中心
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