PFOS替代品对人骨髓间充质干细胞多向分化平衡的干扰作用和分子机制

In view of the problem that the alternatives of perfluorooctane sulfonate (PFOS) pose relatively high environmental health risk with little information on their toxic mechanisms, the present study aims to reveal the mechanism underlying the toxic effects of PFOS alternatives on bone development, based on the imbalance in the pluripotent differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs). The alternatives with short carbon chain or chlorinated polyfluorinated ether group were selected due to the serious environmental pollution, non-biodegradability, and the relatively high bioaccumulation potency. The in vitro culture and induced differentiation system of hBMSCs will be developed. And the patterns, characteristics and the mechanisms of their disturbance effects on the pluripotent differentiation balance in hBMSCs will be studied based on the cell uptake, cytotoxicity, self renewal impairment, and disturbed differentiation potency. The disturbance induced by PFOS alternatives in the phenotype and the molecular biomarker during the dynamic process of osteogenic/chondrogenic differentiation in hBMSCs will be characterized, and its correlation with abnormality in adipogenic differentiation potency will be studied. Furthermore, the cascade regulation of specific genes involved in the osteogenic/adipogenic differentiation balance in hBMSCs, including WNT signaling and PPARγ regulation, etc, will be evaluated to elucidate the interaction of key signaling pathways and transcription factors. In addition, the transcriptomic tools will be employed to explore other potential mechanisms. The results will provide scientific foundation and novel methods for the development of PFOS alternatives with low hazard and toxicity and the environmental health risk assessment.

针对全氟辛烷磺酸(PFOS)替代品潜在环境健康风险较高但毒性机理不清楚的问题，本项目拟基于人骨髓间充质干细胞(hBMSCs)多向分化失衡，揭示其骨骼发育毒性机理。选择环境污染水平较高、难降解且生物累积性较强的短碳链和氯代多氟醚基替代品，建立hBMSCs体外培养和诱导分化模型，从细胞摄取、细胞毒性、自我更新能力损伤和分化潜能紊乱多角度研究其低浓度暴露干扰干细胞多向分化平衡的形式、特点和作用机制。研究PFOS替代品对hBMSCs成骨/成软骨分化表型特征和分子标志物动态过程的干扰效应，并考察成脂分化潜能异常与该干扰效应的相关性。研究PFOS替代品诱导WNT信号通路和PPARγ调控等相关特异基因差异表达的级联调控关系，阐明成骨/成脂分化平衡主要信号通路和转录因子的交互作用，并利用转录组学方法探究其他未知机制。研究结果可为研发低毒低害的PFOS替代品及其环境健康风险评价提供科学依据和新方法。

针对新型全氟及多氟烷基化合物(PFASs)潜在环境健康风险较高但毒性机理不清楚的问题，本项目基于人骨髓间充质干细胞(hBMSCs)增殖和多向分化体外毒理学模型，研究了全氟辛烷磺酸(PFOS)、全氟辛酸(PFOA)及其典型替代品的毒性潜能和机理。. 在人体暴露相关浓度下(nM水平)，PFASs对hBMSCs的基因表达和细胞分化产生干扰作用，说明干细胞分化是PFASs的敏感靶标。PFOS以非单调剂量-效应关系抑制hBMSCs成骨分化并促进其成脂分化，分子水平和表型特征变化一致，结果很好地解释了人群流行病学调查发现PFOS暴露与骨密度降低相关的机理。4种PFASs作用于干细胞的毒理基因组特征显著相关，提示PFASs具有相似的毒性作用模式，其中成骨分化是多种化学品共同干扰的最显著的通路。此外，毒理学基因组学变化和相应的毒性终点测试结果均证实，细胞干性和钙稳态等也是多种PFASs的共性靶标，其中PFOS替代品氯代多氟醚基磺酸(Cl-PFESA)对钙瞬变的影响作用比PFOS更强更持久。进一步基于稀有鮈鲫体内毒性测试，证实了发育期Cl-PFESA暴露对内分泌调控和生长发育的干扰效应。. 基于毒性通路的毒理学机制研究表明，PFASs主要通过WNT/β-catenin、OPG/RANKL/RANK和PPAR信号通路干扰干细胞增殖和多向分化平衡。PFOS和全氟己烷磺酸(PFHxS)主要通过诱导PPARγ表达抑制成骨分化，但替代品Cl-PFESA由于对PPARβ较强的激活作用对成骨分化产生促进作用。与磺酸类化学物对干细胞的毒理基因组学影响不同，羧酸类化学物对细胞增殖和多条癌症相关的通路产生干扰作用，RT-qPCR和Western blot测试证明了PFOA替代品六氟环氧丙烷二聚体铵盐(HFPO-DA)和三聚体(HFPO-TA)影响癌症标志物基因表达，EdU增殖实验证实HPFO-TA促进细胞增殖。. 结果提示人间充质干细胞增殖和多向分化体外模型能够灵敏的检测低浓度PFASs类化学物的毒性潜能，并能够基于毒性通路精准识别化学物特异性的毒性机制。研究结果为PFASs的环境人群流行病学骨骼健康调查结果提供了毒理学机制，为该类化学品的健康效应预测和风险评估提供科学依据和高通量毒性测试技术。