甲基汞对斑马鱼侧线器官细胞分化的毒性机理研究

Mercury is known to be a teratogen, neurotoxicant and to increase risk of cardiovascular disease and hypertension. Mercury is emitted into the environment through normal geological process, volcano eruption and is produced anthropogenically by industrial activities. The increment of mercury emission in environment is observed around world, particularly in China. Due to the widespread prevalence of mercury and its derivatives in the environment, the contact of human with these toxic chemicals is unavoidable. Mercury started to receive special attention after several serious Hg poisonings happened in Japan and Iraq in the last century. Children with maternal exposure to mercury showed extensive damage of the brain, particularly the cerebral and cerebellar cortices. However, we know little about how mercury can influence the neuron development and affect the intelligence. To understand the mechanism of teratogen, my group carried out a systemical study of gene expression profiles in exposure the zebrafish to Methyl mercury (MeHg) by using microarray analysis and in situ hybridization. The results indicated that MeHg caused dysregulation of various biological processes and molecular functions, such as biological function related to cell death, apoptosis, inflammatory response and response to oxidative stress and altered the gene expression in the brain. On the basis of these results, the zebrafish lateral line system is empolyed to understand how the cell migration, neuromast deposit, hair cell differentiation and cell death are affected in MeHg exposure embryos by combining analysis of gene expression profiles and tracing the GFP marked cell migration. Finally, the genes triggered in MeHg treatment are defined in the gene regulated network and the key genes with potentials to compensate the toxic effect of MeHg will be identified. The hair cells in the lateral line have the similar function as the hair cells in human inner ear. The zebrafish lateral line is an important model for investigating the differentation, cell death, regeneration of hair cells. This model will provide more information to understand the mechanism of deafness induced by environmental factors.

汞对人类神经和心血管系统的正常功能具有负面影响。人类的生产活动，不断增加环境中汞的排放量和汞的沉积，扩大了食物链的累积效应，使人类健康不可避免的受到影响。20世纪发生在日本和伊拉克的群体性汞中毒事件，使公众深刻的认识了汞毒性对胎儿、婴幼儿神经系统、智力发育等造成的严重损伤，然而汞如何影响神经系统和智力发育我们目前了解的不多。在我研究组多年、系统研究甲基汞对斑马鱼胚胎基因表达影响，神经发育作用的基础上，本项目拟用斑马鱼侧线器官作为模型，结合基因表达分析、荧光标记技术，研究甲基汞对侧线器官原基运动、毛细胞分化、死亡的影响作用。在此基础上，揭示诱导表达基因的相互作用，构建基因调控网络，发现关键基因和可以消减甲基汞毒性的靶基因。阐明甲基汞影响侧线器官原基运动、毛细胞分化、诱导细胞死亡的分子机理。建立研究环境因素引起毛细胞分化、死亡的动物模型，为揭示环境因素导致人类神经性耳聋的分子机理奠定基础。

本项目通过构建在斑马鱼侧线器官表达荧光的转基因斑马鱼，根据组成侧线器官细胞的荧光强度，观察、记录甲基汞暴露导致侧线器官细胞数量变化并建立浓度效应关系；通过时间间隔电影，动态研究甲基汞对侧线器官迁移、细胞分化过程的影响；通过转录组学分析、揭示甲基汞诱导不同发育阶段斑马鱼胚胎基因表达谱；通过原位杂交、基因敲除或者敲底和基因过表达，揭示在侧线细胞表达，并受到甲基汞影响的关键基因功能与侧线器官细胞分化的关系，在此基础上，揭示甲基汞影响侧线器官细胞分化，进而导致毛细胞死亡的毒性机理。通过以上研究内容，项目取得以下成果：1）甲基汞不影响侧线器官原基的迁移和外套细胞（Mantle cell）、毛细胞分化；2）甲基汞暴露导致斑马鱼侧线器官毛细胞数量减少与其浓度和暴露时间呈正相关；3）甲基汞诱导的基因谱与斑马鱼发育阶段、暴露浓度和时间有关；4)甲基汞暴露诱导72小时和120小时斑马鱼胚胎132个和133基因呈现显著性差异表达；5）基因原位杂交揭示15个差别表达基因在侧线器官表达；6）通过敲低和过表达Thioredoxin1基因，揭示Thioredoxin1功能和侧线毛细胞发育有关。通过该项目，我们首次系统揭示了甲基汞对斑马鱼侧线器官形成和侧线器官毛细胞分化的影响，浓度效应关系，并阐明了甲基汞对侧线毛细胞毒性效应和毒性机理，发现了Thioredoxin1在侧线器官毛细胞分化过程中的重要功能，为进一步深入研究甲基汞暴露导致人类耳聋的致病机理提供了科学数据，同时为建立有效评价甲基汞水生动物健康风险方法提供了新的途径。