多溴联苯醚诱导海洋鱼类生殖细胞凋亡的途径及分子机制研究

It is of great significance to reveal the responses of marine fish reproduction to the global changes, because it's one of the most beneficial ways to help clarifying the possible ecological mechanism of the coastal biological resources recession. Studies on germ cell apoptosis is suggested to be the most direct way to elucidate the reproductive toxicity. The present research thus choose the marine fish's germ cell as the breakthrough point and apply the new type of persistent organic pollutant- polybrominated diphenyl ethers (PBDEs) as the stressing factor, and a serious experiements are designed under controlled lab conditions to find the possible pathway and molecular mechanism. We have found in the previous studies that PBDEs exposure could induce the reproductive toxicity in marine zooplankton, increase the reactive oxygen species (ROS) level in the organism, lead to domino effect of oxidative stress and subsequently result in immune responses. However, no causation relation between the ROS level increment and the reproductive toxicity has been studied. Therefore,the aim of the present research is to answer the following questions: 1. what's the role of reactive oxygen species(ROS) in apoptosis induction by PBDEs ? That means we want to know the ROS level increment is the "reason" or "result" of PBDEs stress? 2. ROS-mediated pathway could be mitochondria pathway, endoplasmic reticulum pathway and the MAPK kinase pathway, and which one is involved germ cell apoptosis induced by PBDEs? Given the questions above mentioned and basing on our preliminary research results, we change the intracellular ROS levels by redox state modulators (NAC, CAT, Tiron and BSO)to find out the causation relation between ROS level and reproductive toxicity induced by PBDEs . To insight the ROS-mediated signal pathway，we examined the expression of stress activated protein kinase (SAPK) phosphor-JNK、phosphor-p38、JNK、p38. To further confirm the PBDEs-induced apoptotic signal pathway, we examined Cyt c, Caspase3, Caspase9, PARP, GRP78, GRP94, CHOP, Caspase7 and Caspase12 by Western Blot. The in vivo toxic analysis in model fish will be performed simultaneously so that more evidences could be applied for evaluating the effectively mechanism of PBDEs' reproductive toxicity. The results of present study would provide research foundations for revealing the effective pathway and molecular mechanism of PBDEs on marine organisms.

阐明鱼类生殖过程对全球变化的响应对于揭示近海生物资源持续衰退的生态环境机制具有重要意义，而生殖细胞凋亡则是分析生殖毒性机理的最直接的方法。本研究因此以海洋鱼类生殖细胞研究为切入点，以全球性新型持久性有机污染物多溴联苯醚(PBDEs)为胁迫因子，将体外细胞毒性与模型动物的体内毒性研究相结合，系统分析PBDEs诱导的生殖细胞凋亡及分子作用机制。我们已经发现PBDEs胁迫过程中通常伴随着活性氧（ROS）升高。为此，本研究采用PBDEs单独或与氧化还原剂联用，通过人为干扰细胞内ROS水平，阐明细胞ROS水平和凋亡的因果关系；通过检测MAPK通路中各元素的变化，阐明生殖细胞凋亡过程中ROS介导的信号通路；通过线粒体途径和内质网途径中标志性指标或元素的变化确定细胞凋亡的具体通路；同时结合动物模型的体内毒性研究，进一步确证毒性作用的途径与机制。研究将为揭示PBDEs生殖毒性的分子机制提供依依据。

新型持久性有机污染物多溴联苯醚(PBDEs)对海洋生态系统存在潜在威胁。本研究将动物模型与细胞检测相结合、将体内与体外毒性效应研究相结合，研究PBDEs诱导的毒性与凋亡作用，阐明可能的作用机制与途径。主要回答以下两个问题：ROS 是毒性与凋亡的“因”还是“果”？海洋低营养级生物应激响应的途径与机制是什么？结果表明：.(1)以小鼠神经瘤细胞Neuro-2a和虹鳟鱼生殖细胞RTG-2为目标的体外毒性实验表明：a. PBDEs具有明显的细胞毒性，诱导细胞产生凋亡，干扰细胞周期与细胞分裂，将细胞阻滞于G1期；线粒体通路、内质网通路和死亡受体通路等通路均参与PBDEs诱导的细胞凋亡过程中。b. Ca2+和ROS的联合作用是细胞凋亡的触发因子。PBDEs胁迫诱导Ca2+和ROS水平升高，并顺序性引起细胞内氧化还原状态变化、诱导p38MAPK通路的活性；Nrf2-ARE信号通路在应对PBDEs胁迫-响应过程中具有重要的防御作用。本研究首次证明参与海洋鱼类生殖细胞凋亡的3条途径，阐明PBDEs诱导生殖毒性的可能决定机制。.(2)以虹鳟鱼幼鱼为目标的体内毒性实验表明：a. PBDEs能够抑制虹鳟生长，头肾和肝脏是PBDEs的主要蓄积部位，提示其免疫系统是PBDEs影响的靶系统。PBDEs通过损伤免疫器官结构、降低免疫细胞功能、影响免疫因子活性、干扰免疫基因表达等途径影响虹鳟鱼的免疫系统，通过干扰非特异性免疫能力而降低其对对外源有害细菌的抵御能力。b. BDE-47胁迫能上调头肾核受体PXR表达，并顺序激活I相、II相解毒酶及其编码基因的表达活性，增强机体代谢解毒能力；当蓄积的PBDEs超过其解毒能力时则造成免疫器官的氧化损伤。代谢解毒系统和抗氧化系统损伤使得以头肾为代表的免疫器官/组织的免疫功能下降，这是BDE-47诱导虹鳟幼鱼免疫毒性的重要原因之一。.本研究首次实现高等动物中的普适性研究方法在低等海洋动物中的应用，为进一步拓展海洋环境生态学领域的效应与机制研究提供新思路。基于本研究，共发表学术论文17篇，授权专利2项，申请发明专利3项，过得省部级奖励1项。