低浓度有机磷农药胚胎脑毒性的顶底极性及三维干细胞发育模型

One of the major concerns for ecological safety is the low but persistent exposure of widely used pesticides like chlorpyrifos in ordinary life. It was proved that developing brain as a susceptible window of organophosphates exposure, which may elicit long time damages in neural tissue structure and functions. The mechanism involves multiplication, differentiation, and apoptosis of neural progenitors. Transition from self-renew to differentiation of embryonic neural stem cell is a key event of neurogenesis, which progression with switching of symmetric/asymmetric division, lengthening of cell cycle, polarization of the cells, and evolution of developmental markers. The dynamic balance is fragile to environmental factors. Our previous results indicate organophosphates as disruptors in the balance. Apical-basal gradient of cytoarchitecture and developing markers in embryo neocortex represents the transition procession, suppling an idea model for our study. In this project, we plan to investigate the effects of low concentration organophosphates on cell stratification, apical-basal ultrastructure, developmental markers, and Wnt/shh gradient, using embryonic neocortex slices combined with multiple immune fluorescence and immune electron microscopy techniques. Moreover, we will set up a 3D stem cell development model on these study results. This project based on our previous works, investigates neural developmental toxicity mechanism of low concentration organophosphate in a novel angle, and is possible to develop a new evaluation method.

常用有机磷农药如毒死蜱在日常生活中的低浓度长期暴露问题构成重要的生态安全隐患。胚胎神经发育期是低浓度有机磷农药毒性作用的敏感暴露窗口，可造成脑结构机能持久性损伤，其作用机制涉及发育期细胞的增殖、分化与凋亡。胚胎神经干细胞自我复制/分化转换是神经发育的源头,经历对称/不对称分裂转化、细胞周期时程渐变、细胞极性发生、发育标志衍变等一系列过程,其动态平衡对环境因素敏感。我们的前期工作已揭示低浓度有机磷农药干扰这种转换。本项研究以胚胎新皮层脑室壁顶底极性为发育模型，深入探讨低浓度有机磷农药对胚胎神经干细胞自我复制/分化转换的影响及其作用机制。拟采用胚胎脑切片结合多重免疫荧光及免疫电镜技术，观察分析低浓度有机磷农药对胚胎新皮层细胞分层、顶底极性超微结构、细胞发育标志以及Wnt/shh信号梯度的影响，并进而建立三维干细胞体外模型。本项目从一个新的角度探讨有机磷农药神经发育毒性，有可能建立新的评价方法。

研究结果发现：（1）低浓度毒死蜱直接作用于顶端神经前体细胞分裂环节，抑制细胞分裂进程并延缓细胞周期运转，这种作用在停止暴露农药后仍持续存在；（2）有丝分裂平面取向改变仅涉及表层神经前体细胞而不影响非表层神经前体细胞，并且此时细胞并未发生凋亡、核质凝聚、胞内空泡等病理性改变；（3）对细胞分裂增殖的抑制作用仅影响顶端部的源头神经前体细胞，而对脑膜面已经分化的神经元影响不大甚至起轻度刺激作用；（4）由于脑组织中未分化、低分化、已分化神经前体细胞比例失衡，胚胎侧脑室壁的细胞分层结构发生异常；（5）低浓度有机磷农药抑制体外培养的胚胎神经前体细胞神经球聚集。这些研究结果揭示了低浓度有机磷农药作用下，胚胎神经前体细胞从分裂模式转换异常到脑组织结构构建异常的病理过程。