体外三维培养肝微组织研究PFASs分子结构与肝毒性的关系及分子机制

Per-/ployfluoroalkyl substances (PFASs) are persistent, bioaccumlative compounds with toxicity. Their potential environmental and ecological hazard, as well as on human health has led to a great concern. PFASs include many sorts of compounds with different functional groups and carbon-chain length. Animal studies suggest that the liver is a primary target organ for PFASs physiological activity. However, different sorts of PFASs do not exert their biological effect with the same mechanism, and the most sensitive molecular is not identified yet. Based on our previous studies, we plan to obtain hepatic microtissue with three dimensional cell culture technique, and use the microtissue to perform toxicological research on cell cycle progression, stress and metabolism, and to identify the common points and difference between PFASs with different functional groups and carbon-length; to obtain the effect of PFASs on key toxicological signal pathways, and identify the important molecular contributors to the hepatotoxicity, as well as the mechanism of species difference. In addition, using hepatic microtissue obtained by coculture of hepatocyte, Kupffer cell and hepatic stellate cell, we will study the intercellular communication and the function of secretory cytokines. By these studies, we will identiy the most sensitive molecular and biomarkers for PFASs exposure and effect, identify the molecular mechanism behind the hepatotoxicity of PFASs, and obtain scientific data which will be helpful to the hazard control and regulation of PFASs.

全/多氟烷基化合物(PFASs)具有持久性、生物累积性和毒性效应，其对生态环境和人体健康的潜在危害广受关注。肝脏是PFASs的重要效应器官，PFASs因碳链长度和功能团的差别有多种类型，但不同类型PFASs发挥肝毒性效应的机制不尽相同，而且尚不确定PFASs毒性的具指示性敏感指标。结合前期的工作基础，本项目拟通过体外三维立体细胞培养方式获取肝脏微组织，利用肝微组织模型研究PFASs不同功能基团和碳链长度对肝脏增殖、应激与代谢等生物效应的影响，确定主要PFASs类型生物效应的差别与共性；确定PFASs代表性化合物对毒性信号通路的作用、敏感指标分子及其物种差异性分子基础；通过肝实质细胞与肝巨噬细胞及肝星状细胞组合共培养研究细胞间信号分子，炎性因子在PFASs肝毒性中的作用。该研究有望揭示PFASs肝毒性的分子基础，为评估PFASs的健康影响和制定PFASs管理策略提供科学依据。

PFOA有稳定而强劲的C-F键，环境和健康危害广受关注。3D悬滴培养技术采用无支架介质形成3D微组织，更接近体内生理环境。我们利用小鼠肝脏细胞系AML12制作3D悬滴微球，比较了传统2D培养与3D微球对PFOA的反应差别，发现相对于单层细胞，3D细胞具有更强的耐受性。PFOA暴露3D微球至少能存活4周，其白蛋白分泌、ATP含量及LDH释放等评价均支持3D微球可满足长时间培养和毒性评估需求，是一个好的毒性效应体外评估系统。进而使用鼠肝脏组织获取的细胞混合物制作了更贴近生理实际的3D微组织，比较研究了PFOA和新型替代品HFPO-TA的毒性，发现HFPO-TA更易在肝微球内累积，Albumin分泌、ATP含量及LDH释放均支持HFPO-TA具有比PFOA更强的肝脏毒性效应。微组织RNA-seq分析表明两个化合物暴露引起胆固醇代谢、炎症和胆汁酸代谢异常；微球中总胆汁酸水平升高，胆汁酸运输相关的基因Bsep和Mrp2的表达显著下降，HFPO-TA比PFOA长期暴露更能引起微球内发生胆汁淤积。总之，3D微组织评估结果表明HFPO-TA毒性较PFOA更大，不是理想的替代品。