环境内分泌干扰物对模式鱼免疫系统的毒性效应以及作用机理研究

It is now firmly established that the presence of environmental endocrine disrupting chemicals (EDCs) in the aquatic system results in the decline of fish population. The cause of this phenomenon may not only attribute to the impairment of reproductive axis of exposed fish, but also the disruption of their immune competence and, consequently, their resistance against infections. However, most of the currently existing data only focus on the descriptions of effects, which is too limited to be conclusive for the mechanisms involved or the range of target processes and functions within the immune system. In this project, with the aid of mechanism for sex identification of Japanese medaka, firstly, we will characterize the ontogenetic and tissue-specific changes in the expression of the immune-related genes, as well as the maternal transfer of immune factors, by using qRT-PCR, in situ hybridization and other immunological assays. Secondly, the impacts on the immune system of different life stages and the maternal transfer of medaka exposed to several EDCs targeting the hypothalamic-pituitary-gonadal (HPG) axis will be investigated systematically. The different immunotoxic effects between the resting and activated immune systems in fish for EDCs will be assessed as well. Moreover, the role of sex steroid hormone receptors on immunomodulation will be studied. Finally, the transcriptional changes of a set of genes involved in the HPG axis of medaka will be examined after challenge with lipopolysaccharide, and the feedback of immune system to endocrine function will be explored. This project will not only provide useful information for the ecological hazard assessment of EDCs, but also have important implications for the understanding of bi-directional communications between the endocrine and the immune system.

研究发现环境内分泌干扰物的暴露将降低鱼类种群密度。这不仅可归因于其对鱼体内分泌系统的损害，也可能是通过扰乱免疫功能从而降低其对感染的抗性所导致。然而，环境内分泌干扰物对鱼类免疫系统的影响研究多集中在效应的描述上，而对靶标和机理的确定还缺少明确结论。本课题借助模式鱼青鳉的性别决定机制，通过定量PCR、原位杂交等技术及其他免疫学指标，在明确雌雄青鳉不同发育阶段免疫相关基因转录水平变化、组织分布以及免疫因子母源性传递的基础上，通过靶点在HPG轴上的多个典型内分泌干扰物的单一和共暴露，系统研究其对不同生命阶段、不同免疫状态下青鳉的免疫毒性，并明确性激素受体在免疫调节中的作用。同时，通过研究免疫刺激后青鳉内分泌HPG调控轴关键基因的转录变化，确认免疫系统对内分泌系统的反馈作用。本研究不但为准确评价环境内分泌干扰物的生态毒性提供科学依据，也为阐明内分泌系统和免疫系统间的双向信息交流机制打下良好基础。

环境内分泌干扰物不仅可以直接损害鱼类的内分泌系统，而且可通过扰乱其免疫功能，从而降低其对感染的抗性，最终导致其种群密度下降。然而，我们对于有关环境内分泌干扰物对水生生物的免疫毒性，以及鱼类内分泌系统和免疫系统间相互关系等还认识有限。针对上述问题，本项目借助于青鳉等模式鱼，较为系统地评价了典型内分泌干扰物对于模式鱼免疫系统的影响。首先，建立模式鱼青鳉单个胚胎和幼鱼的总RNA和基因组DNA的同步提取策略，优化了以dmrt1bY基因鉴定青鳉鱼性别的方法。其次，完成了模式鱼青鳉早期生命阶段的二代转录组测序以及数据分析，较为全面的阐明了青鳉早期发育阶段的基因变化以及性别差异，并针对免疫系统相关基因，通过qRT-PCR验证了其转录水平的变化，从而明确了免疫系统在青鳉不同阶段的发育状况，确认了硬骨鱼免疫系统的性别差异。再次，以雄激素群勃龙（TRB）和雌激素乙炔基雌二醇（EE2）为典型的环境内分泌干扰物，研究了青鳉在生命早期阶段暴露于TRB和EE2后，免疫系统相关基因的转录水平变化，初步确定了雄激素和雌激素等典型内分泌干扰物的免疫毒性，并且毒性存在显著的雌雄差异。同时，以LPS为免疫刺激剂，对比了免疫系统在激活或者静息状态下，雌激素暴露对于模式鱼斑马鱼免疫系统的不同影响。最后，鉴于免疫系统在组织修复和再生中的关键作用，通过斑马鱼尾鳍再生模型，研究了免疫系统强烈激活的情况下，雄激素和雌激素暴露对断尾再生的影响。以上研究不但系统评价了环境内分泌干扰物对于硬骨鱼的免疫毒性，确证了内分泌系统对于免疫系统的影响，而且表明了免疫系统对于内分泌系统的反馈调节作用，从而为阐明内分泌系统和免疫系统间的双向交流机制提供了科学依据。