模拟微重力下斑马鱼先天免疫研究模型建立及其对抗病毒I型干扰素系统影响机理研究

Microgravity is considered as the major environmental factor of space flight that affects immune system causing adverse effects to astronaut’s health. Ground-based gravity-simulation experiments have gained some insights into the underlying molecular and cellular modification induced by microgravity. However, systematic study and detailed molecular mechanisms of the adverse effects of microgravity on the immune system of living animal models are still limited. To this end, this project contributes to the establishment of a novel zebrafish model for the research of innate immunity in space, as well as the elucidation of the molecular and cellular mechanisms of disorder of type I interferon (IFN) signaling in microgravity. The rotary cell culture system (RCCS) is applied to create ground-based modeled microgravity condition, and DNA microarray technology is used for genome-wide search of innate immune genes whose expression are altered by microgravity. Further, the variation of genes involved in type I IFN signaling in response to the microgravity will be focused. To reveal the continuous state of type I IFN signaling under the microgravity environment, the kinetic expression patterns of its candidate genes during the time-cause microgravity treatment are sought to be analyzed. Besides, the analyses on the expression variation of the candidate genes under microgravity condition upon stimulation by different virus signals will to be executed, which may show the varied responses of type I IFN signaling to different types of viruses in the microgravity condition. Moreover, we attempt to investigate the molecular and cellular mechanisms of the effects of microgravity on host antiviral regulatory network. By using the technologies of gene knock-down and overexpression, the expression of candidate genes in type I IFN signaling are to be modified in zebrafish embryos, and whether these modification can rescue the function of type I IFN signaling in responses to virus signals under microgravity condition will be determined. By using the dual-color fluorescence reporter gene system, we intend to in situ observe the varied expression of the key candidate genes in monocytes/macrophages under microgravity condition, as well as to track their migration into the viral component injection site. Through these investigations, we hope to establish a novel and efficient animal model for the research of space flight immunology, screen the innate immune-related genes underlying the systemic regulatory networks in response to microgravity, and reveal the mechanisms of the disorder of type I IFN signaling under microgravity condition. We wish the results of this project will not only provide new insights into better recognition of the effects and mechanisms of microgravity on host antiviral immunity, but also has important application potential in the development of strategies in prevention and control of host diseases against virus infection under space flight.

微重力环境对机体免疫系统的影响是航天生物效应研究的核心问题之一，严重影响了航天员在太空旅行中的健康和任务执行能力。本项目拟基于DNA芯片等技术，建立旋转细胞培养系统（RCCS）模拟的微重力环境下先天免疫斑马鱼胚胎研究模型，全面筛选和分析斑马鱼胚胎中响应微重力环境的先天免疫相关基因，并重点研究I型干扰素（IFN）系统候选基因表达和对病毒信号响应的情况。进一步结合分子和细胞实验，深入探索微重力引发的I型IFN系统紊乱的分子机理，以及对单核/巨噬细胞表达I型IFN及其向病毒侵染部位迁移能力的影响。该项目将为航天微重力免疫效应的研究和相应防治措施的开发提供新的思路和理论基础，为我国正蓬勃发展的航天事业提供重要保证。

航天微重力生物学效应及机制的研究一直是空间生物学研究领域核心的科学问题之一，严重影响了航天员在太空旅行中的健康和任务执行能力。目前微重力条件对免疫功能影响的效应和机制研究尚处于起步阶段。本项目基于RNA-seq等技术，以RCCS模拟太空微重力效应，全面筛选和分析了斑马鱼胚胎中响应微重力效应的先天免疫相关基因，建立了用于研究模拟微重力效应下先天免疫应答的斑马鱼胚胎模型。重点分析dsRNA病毒模拟物poly I:C刺激结合模拟微重力效应处理前后斑马鱼胚胎I型IFN信号系统活化情况，发现模拟微重力效应下机体I型IFN诱导通路（TLR3和RLR信号通路）和I型IFN下游效应通路（JAK1-STAT1/2信号通路）均发生了强烈的抑制。重点筛选上述通路差异表达的关键基因，发现TRIM25的表达降低可能是影响微重力效应下I型IFN表达受到抑制的重要因素。通过在斑马鱼胚胎中过表达或敲低TRIM25，证明了微重力效应下TRIM25低表达是造成I型IFN信号通路受阻的原因之一。利用免疫沉淀法和定量PCR等技术分析斑马鱼胚胎中TRIM25活化I型IFN信号通路的功能机制，发现TRIM25通过调节RIG-I 分子K63连接多聚泛素化影响IFN信号通路活化，而该机制正是微重力效应下TRIM25低表达干扰I型IFN信号通路的主要原因。此外，研究发现机体直接过表达I型IFN分子无法完全拯救IFN诱导基因的表达，而JAK1-STAT1/2信号通路的STAT1和Tyk2基因在微重力效应下的低表达是I型IFN效应通路受阻的关键因素。本研究初步阐明了微重力引起机体抗病毒免疫功能紊乱的潜在分子机理，为航天微重力生物学效应的研究提供新的思路和理论基础，对于获得太空微重力对机体免疫功能影响的足够信息，明确微重力环境下机体免疫功能紊乱的具体机制，并开发出相应的防护措施具有的潜在理论和应用价值，对于我国正蓬勃发展的航天事业具有重要意义。