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基于胚胎干细胞技术的二恶英等污染物发育毒理学研究
Alternative TitleHuman embryonic stem cell derived developmental toxicology study on dioxin and other environmental pollutants
付华龄
Subtype博士
Thesis Advisor赵斌
2019-06
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline环境科学
Keyword胚胎干细胞,二恶英,发育毒理,心肌发育,中胚层 embryonic Stem Cell, Dioxin, Development Toxicology, Cardiomyocyte Induction, Mesoderm
Abstract

      环境污染物是人类健康的一大威胁,很多环境污染物被证明具有发育毒性,其中二恶英是一种公认的致畸物,对包括心血管、骨骼、肝脏、牙齿、生殖系统
等多种器官与组织的发育都具有毒性作用。一些新型污染物的发育毒性也逐渐被
发现。传统的发育毒理学研究通常借用动物模型,例如斑马鱼、非洲爪蟾、鸡胚
与小鼠,或者使用具有一定分化能力的癌细胞。但由于种属差异和癌细胞的组织
特殊性与分化能力局限性,这些方法并不是对人类健康最好的参照。近年来,胚
胎干细胞技术飞速发展,在基础生物学和再生医学研究中发挥了重要的作用,也
为发育毒理学研究提供了理想的工具。与此同时,新的分子生物学技术如
CRISPR-Cas9 基因编辑,高通量测序等也为毒性机理的研究提供了强大的辅助。
      本研究基于人胚胎干细胞技术和一系列细胞生物学与分子生物学技术构建了基于人胚胎干细胞技术的发育毒理学研究平台,即利用人胚胎干细胞体外分化的过程模拟特定器官或组织在环境暴露下的发育过程,确定污染物的发育毒性和敏感阶段,再结合信号通路研究、组学技术等对毒性机理进行探索。
      我们对二恶英的心肌发育毒性研究进行了系统和细致的研究,弥补了二恶英对人类心肌发育毒性实验研究的空白。在这一研究中,我们首先通过对发育过程
的全程暴露证实了2nM 二恶英(2,3,7,8-TCDD)对人胚胎干细胞的体外心肌发育具有毒性作用,导致心肌、离子通道、房室结等组织标志物表达量降低,心肌细胞形态异常。接着,我们通过分发育阶段的暴露实验找到了心肌发育对二恶英毒性最敏感的发育阶段为发育早期的胚胎干细胞阶段和中胚层发育阶段。之后,我们通过芳香烃受体AhR 的拮抗和CRISPR-Cas9 敲除,对AhR 信号通路在毒性
介导中的作用进行了研究,证实了二恶英对心肌发育的毒性是通过AhR 信号通
路介导的。最后,我们借助染色体组免疫沉淀测序、转录组测序,和生物信息学
分析手段,发现AhR 可以直接结合并调控一系列中胚层关键性基因,从而影响
后续的发育过程。
      根据我们在二恶英心肌发育毒性研究中的一些新发现,例如AhR 信号通路在人们熟知的毒性介导作用以外,可能在发育中具有复杂的调控作用,我们对AhR 信号通路与胚胎干细胞全能性的关系进行了探索,发现AhR 信号通路对胚层分化与干细胞的DNA 甲基化都会造成显著影响,表明AhR 信号通路对人类胚胎干细胞全能性可能存在调控作用。我们也进一步对类二恶英类物质,如近年来受到关注的咔唑类污染物,进行了发育毒性研究。我们发现,具有最强的类二恶英效应的2,3,6,7-氯代咔唑暴露对经典二恶英毒性通路的激活量较小,但却会对中胚层发育造成较强的抑制。
      综上,本研究将最新的生物技术应用到环境领域,建立了一套新的发育毒理学研究方法,对热点的环境问题进行了研究,得到了有价值的数据,证实了该研究方法在环境毒理学研究方面的能力,并通过方法和研究对象的拓展证实了胚胎干细胞在毒理乃至基础生物学研究中的潜力。

Other Abstract

      Environmental pollutants are a major threat to human health. Many environmental pollutants are proved as developmental disruptors. For example, dioxins are known as a strong teratogen which could cause various developmental toxicity including osteogenesis, cardiovascular genesis, liver development, tooth development, and reproductive system development. Developmental toxicity of newly discovered environmental chemicals also draw people’s attention.
      Based on human embryonic stem cell (hESC) technology and the most advanced molecular biology technics such as CRISPR-Cas9 genome editting, we developed a novel developmental toxicology study platform which use the induced in vitro hESC differentiation to mimic the in vivo develop process of human organ or tissue under environmental pressures. Using this platform, we can not only verify the developmental toxicity of environmental chemicals in human system, but also find the sensitive
develop stages and study the mechanism with the aid of pathway studies and omics data analysis.
      This hESC derived developmental toxicology study method have been
successfully applied on dioxin’s cardiogenesis toxicity study which filled the gap between epidemiology reports and animal experiment data. In this study, we (1) verified the cardiogenesis toxicity of 2nM dioxin (2,3,7,8-TCDD) exposure in human cells; (2) found the most sensitive development stages which is embryonic stem cell stage and mesoderm development stage; (3) discovered the AhR-dependent inhibition effect during the mesoderm commitment and found the regulatory effect of AhR on mesoderm master genes; (4) had shown the power and potential of this novel developmental toxicology study method.
      By application of multiple differentiation protocols, hESC can be easily adapted to the study on other lineages. Since evidence have already shown that AhR have regulatory role in mesoderm development, we adopted the endoderm and ectoderm differentiation methods and DNA methylation sequencing to study the function of AhR signaling pathway on stem cell potency. It is found and activation of AhR signaling pathway inhibited the mesoderm and endoderm differentiation and altered hESC DNA methylation. We have also used this method to screen newly discovered environmental pollutants such as halogenated carbazole. In this study, we found that 2,3,6,7-Tetrachlorocarbazole has significant inhibition effect on mesoderm commitment with a low AhR-activation dose.
      Above all, this study adopted the state of art biological technics into the study of environmental chemicals and built up an advanced hESC based developmental toxicity research method. This method not only enables systematic dissection of the human developmental toxicity of environmental chemicals but also have potential on fundamental biology research and chemical toxicity screening.

Pages130
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/42197
Collection环境水质学国家重点实验室
Recommended Citation
GB/T 7714
付华龄. 基于胚胎干细胞技术的二恶英等污染物发育毒理学研究[D]. 北京. 中国科学院生态环境研究中心,2019.
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