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基于胚胎干细胞模型研究环境污染物对神经肝脏发育的干扰效应
Alternative TitleStudy the developmental neural and hepat ic toxicity of environmental pollutants with embryonic stem cells
梁胜贤
Subtype博士
Thesis AdvisorFrancesco Faiola
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环境污染物, 胚胎干细胞, 神经发育 毒性,肝 脏发育 毒性 Environmental Pollutants, Embryonic Stem Cells, Developmental Neural Toxicity, Developmental Hepatocytes Toxicity
Abstract

      近几年随着环境科学的发展,环境污染物的健康风险效应成为大众关注的热点话题。与成体不同,在胚胎发育过程中,外界因素的轻微扰动会致使胎儿发育发生变化,严重的可能产生致畸效应。特别是 神经系统和肝脏 组织,经证实 是环境污染物的主要作用靶点。神经系统因其发育时间长、发育过程复杂,因此 最 易受外界因素干扰;肝脏 是人体主要的解毒和代谢器官, 往往由于多种化合物的蓄积效应而致使肝脏毒性的发生 。 鉴于许多化合物 可 透过胎盘屏障,威胁胚胎发育,因此探究环境污染物 对神经系统发育和肝 脏 发育 的干扰效应有助于全面评价污染物的健康风险。鉴于此,本文 主要开展以下研究内容:
      (1)探究了 普遍存在 和 广泛使用 的 两种 邻苯二甲酸 酯 类物质( 邻苯二甲酸二乙酯 DEP 邻苯二甲酸二丁酯 DBP 对早期神经系统发育的影响 。结果表明DEP 和 DBP 都可降低小鼠胚胎干细胞 的 细胞活力, IC 50 分别为 1.0 ± 0.3 mM 和0.26 ± 0.05 mM 。 但 DEP 和 DBP 引起细胞毒性 机制不 完全相同 。 DBP 可激活细胞内 caspase 3/7 酶活性、增加细胞膜通透性并引起细胞内 ROS 累积;而 DEP 仅引起细胞内 ROS 累积。 急性毒性 实验 表明相同浓度下 DBP 比 DEP 毒性更强。低剂量(即无 急性 细胞 毒性浓度 10 μM )的 DEP 和 DBP 处理均 可干扰早期胚胎发育,影响三胚层相关基因的表达。拟胚体分化 实验 中, DEP 和 DBP 处理干扰了内胚层( Gata6 、 Foxa2 )、中胚层 T 、 Hand1 )和外胚层 Fgf5 、 Otx2 )标志 基因的表达,且对神经外胚层( Pax6 、 Nestin 、 Sox1 、 Sox3 )的影响最显著。神经前体细胞诱导 实验 进一步证实了 DEP 和 DBP 对早期 神经系统发育的干扰效应。 以上 结果 为 DEP/DBP 暴露影响儿童行为等人群调查结果提供了 细胞分子层面证据 ,并 提示 我们 未来需要 更多关注邻苯二甲酸类物质的健康风险。
      (2) 除邻苯二甲酸类物质外, 卤 代阻燃剂也是 工业生产中 应用 极其 广泛的一大类物质 。 本文探究了该类物质对神经系统发育的影响,并 揭示 五种化合物复合暴露对神经系统发育的影响。转录组 测序 发现 此类化合物影响早期神经发育过程,且可能干扰 轴突 生长 导向及神经元传递等过程 另外, 此类化合物可能通过介导 WNT 信号通路 和 AHR 信号通路 而影响神经系统发育。复合暴露毒性不低于单一化合物暴露,毒性作用更加复杂。BDE 209 的毒性作用 与 BDE 47 相似其潜在的健康风险值得进一步关注; 作为 TBBPA 替代物的 TBBPS 和 TCBPA在相同剂量浓度下,毒性作用均大于 TBBPA ,提示该两种化合物的安全性需格外关注。
      (3) 利用人胚胎干细胞肝分化模型, 探究体外诱导 模型对 甲状腺激素 THsT3 和 T4 )暴露的响应。研究发现分化过程中暴露 T3 和 T4 会诱导形成功能异常的肝脏细胞,表现为糖原储存能力减弱、脂肪异常累积 以及 AFP 、 ALB 、 HNF4A 、APOA1 、 PPARA 、 CYP3A4 、 CYP2C9 等功能基因表达水平 异常 。转录组数据显示 T3 和 T4 显著影响了上千个肝细胞发育和肝细胞功能相关的基因 表明该过程对 THs 处理 极 其敏感。肝细胞诱导早期, THs 处理主要影响肝脏细胞的早期分化信号通路;而肝细胞诱导晚期, THs 处理显著影响 细胞 代谢通路,包括脂肪代谢相关通路、氨基酸代谢相关通路、药物代谢通路、化合物致癌通路、 ABC载体通路等。通过 PPARA 激动剂 GFT505 处理可一定程度上减弱 T3 暴露对肝分化的调控作用, 如 GFT505 处理组脂代谢相关基因( PPARA 、 APOA1 、 APOA2 、PPARR 、 APOA4 、 FABP7 、 APOA3 等)的表达水平更 接近 对照组, 表明该通路在THs 介导的肝发育毒性中有重要作用,为 解毒 提供了一些思路和 科学 依据。
      (4 )从 THs 样品转录组数据中筛选与肝发育和功能相关的差异基因,这些基因对 T3 和 T4 处理响应敏感,可作为 标志 基因用于鉴定和筛选 THs 类似物。共 筛选 得到 69 个潜在的 标志 基因, 并已 通过两个已知的 THs 类似物( BDE 47和 BDE 209 )进行验证。在肝分化过程中暴露 10 nM 、 100 nM 、 1 μM 的BDE 47/BDE 209 分化第 19 天收样测定这 69 个基因的表达水平。结果显示,与对照相比,其中 25 个基因被至少一种 BDEs 显著干扰 这 25 个基因与化合物致癌、类固醇合成、 P450 酶药物代谢等通路相关。有 17 个基因对 BDE 47 和BDE 209 均显 著响应,包括: APOA1 、 AHR 、 PROX1 、 RXRA 、 CYP2C9 、 CYP2C8 、HNF4A 、 CYP1A1 、 CYP3A7 、 THRA 、 THRB 、 PPARA 、 PROZ 、 CYP19A1 、 GNMT 、HSD3B1 、 HPD 。另外,该 实验 结果也证实了 BDE 47 和 BDE 209 对肝发育的干扰效应。这些筛选验证的标记基因可用于测定其它未知污染物是否具有肝发育毒性。
      综上所述 ,本论文利用胚胎干细胞分化模型探究了 多种 环境污染物对神经系统和或 肝脏发育的毒性效应。邻苯二甲酸 酯 类物质( DEP 和 DBP )、 卤 代阻燃剂类物质( BDE 47 、 BDE 209 、 TBBPA 、 TBBPS 、 TCBPA 对 早期神经系统发育 干扰的影响研究 ,为评价和充分认识这类物质的潜在健康风险提供了直接的毒理学数据。基于人胚胎干细胞分化模型,探究了甲状腺激素( T3 和 T4 )暴露在肝分化过程中的干扰效应,以此数据为基础,筛选得到一 批用于评价和鉴定未知污染物肝发育毒性的 标志 基因;进一步通过 BDE 47 和 BDE 209 验证了这些 标志 基因的适用性,为评价未知化合物在肝发育过程中的干扰效应提供参考和指导,具有重要的科学意义。

Other Abstract

      The nervous system and liver are two major targets of environmental pollutants.The nervous system requires years to fully development and performs sophisticated biological processes T hus subtle interference from exogenous chemicals may induce severe developmental neural toxicity. The l iver is the organ responsible for many essential functions including the metabolisms of drugs and xenobiotics, and therefore is the targets of many environmental pollutants. Accumulating evidence has shown that a number of pollutants can permeate the placenta barrier and potentially affect  embryonic development To comprehensivly assess the health risks of environmental pollutants, it is cruci al to consider the effects on the development of the nervous system and liver cells, such as hepatocytes. However , up to now, limited information is available on this topic . Therefore, we performed the following major aims
      (1) Study t he effects of two wid ely used phathalates (DEP and DBP) on the early stages of the nervous system development using mouse embryonc stem cells (mESCs). We showed that both DEP and DBP could decrease mESC viability, with IC 50 of 1.0 ±0.3 mM and 0.26 ± 0.05 mM, respectively. Mec hnistically, DBP induce d intracellular ROS accumulation, cause d cell membrane damage and activate d the caspase 3/7 enzymes, while DEP only induced ROS accumulation. These data suggest that DBP has a more robust acute toxicity than DEP . Moreover, l ow non cytotoxic
concentrations of DEP and DBP (10 μM) might disrupt the early stages of embryo development by dysregulating the expression of markers of three primary germ layers.In fact, during mESC spontaneous differentiation , DEP and DBP disrupt ed the expression of genes responsible for the development of endoderm ( Gata6 , Foxa2 ),mesoderm ( T Hand1 ) and ectoderm Fgf5 , Otx2 ), and significantly altered neural ectoderm markers ( Pax6 , Nestin , Sox1 , Sox3 ). Neural progenitor cells induction assay further confi rmed the potential toxic effects of DEP and DBP on the development of the nervous system. The results can help better understand the associations between phathalates exposure and health problems, and most significantly remind us of the importance of addit ional health risk tests for these two largely used chemicals.
      (2)Assess the effects of five halogenated flame retardants (BDE 47, BDE 209,TBBPA, TBBPS, TCBPA) on the nervous system w ith human embryonic stem cell differentiation assay We utilized RNA seq and qRT PCR analys e s and found out that these chemicals disturb ed the neural development process, and may affect the axon growth/ guidance , neuron transmission processes . In addition, these flame retardants may dysregulate the WNT and AHR signaling pathway s. Interestingly, BDE 209 showed similar tocicity with BDE 47 , whereas TBBPS and TCBPA may not be safe alternatives to TBBPA.
      (3)Evaluate the effects of thyroid hormones (THs, T3 and T4) during the specification of h epatocytes from human embryonic stem cells. We demonstrated that TH exposure yielded to dsyfuntional hepatocytes, with impaired glycogen storage ability, lipid droplets accumulation and abnormal expression levels of AFP , ALB ,HNF4A , APOA1 , PPARA , CYP3A4 , C YP2C9 , etc. Transcript omics analys e s identified over a thousand of differentially expressed genes (DEGs) in samples treated with 30nM THs , s uggesting the hepatocyte differentiation process is very sensitive to THs.TH treatments mainly affected signaling pathways at late differentiation stage s , and dysregulated metabolism and chemical respnding biological processes, including “cholesterol metabolic process”, “lipoprotein metabolic process”, “cholesterol homeostasis”, “glycine, serine and threonine metaboli sm”, “drug metabolism cytochrome P450”, “carbon metabolism”, “fat digestion and absorption”, “PPAR signaling pathway”, “ABC transporters”, etc. Furthermore, the activation of the PPAR signaling cascades with an agonist of PPAR  (GFT505) attenuate d T3 dep endent dysregulation of lipid relative genes PPARA , APOA1 , APOA2 , PPARR , APOA4 ,FABP7 , APOA3 , during hepatocyte differentiation
      (4) Establish a set of biomarkers to screen the developmental hepato toxicity of TH analogues Among the all DEGs related to TH actions during hepatocytes differentiation and function s, we selected 69 of them as potential biomarkers. To validate these biomarkers, we used two famous TH analogues (BDE 47 and BDE 209)and treated ifferentiating cells with various concentrations (10 nM 100 nM 1 μM)of these two chemicals. After 19 days of hepatic differentiation, 25 out of the 69 potential biomarkers were responsive to at least one BDE. These 25 genes were
enriched in biological processes including “ chemical carcinogenesis”, “drug metabolism cytochrome P450”, “steroid hormone biosynthesis”. 17 genes were responsive to both BDE 47 and BDE 209, and includ ed APOA1 , AHR , PROX1 , RXRA ,CYP2C9 , CYP2C8 , HNF4A , CYP1A1 , CYP3A7 , THRA , THRB , PPARA , PROZ ,CYP19A 1 , GNMT , HSD3B1 and HPD T hus, these genes may serve as biomarkers for the prediction of unknown developmental hepatotoxicants
      In conclusion, we employed embryonic stem cell based differentiation assays to evaluate the effects of environmental pollutants on the development of the nervous system and the most aboundant type of cells of the liver, hepatocytes. Both phathalates (DEP and DBP) and halogenated fl ame retardants (BDE 47, BDE 209,TBBPA, TBBPS, TCBPA) may disrupt the development of the nervous system. These results provide direct evidence for the compre he nsive understanding of the potential health risks of these chemicals. Furthermore, by employing t hyroid hormones ’treatment during human embryonic stem cell differentiation assay s , we identified a set of biomarkers to predict unknown hepatotoxicants. These biomarkers were validated with two toxicants, BDE 47 and BDE 209, providing a reference for the screening of
other chemicals.

Pages161
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/42245
Collection环境化学与生态毒理学国家重点实验室
Corresponding Author梁胜贤
Recommended Citation
GB/T 7714
梁胜贤. 基于胚胎干细胞模型研究环境污染物对神经肝脏发育的干扰效应[D]. 北京. 中国科学院生态环境研究中心,2019.
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