内质网应激介导微囊藻毒素诱导草鱼肝细胞凋亡的机制及GRP78的调控研究

As the largest fresh water lake in china, Poyang Lake is being threatened by the progressive eutrophication of water bodies. The occurrence of cyanobacterial bloom can produce microcystins which is highly hepatotoxic.Induction of apoptosis is one of the important mechanisms of its toxicity, however, the apoptosis pathway of endoplasimic reticulum stress (ERS) is still unkown. On the base of our research, ERS was observed in hepatocyte from grass carp, and some other apoptosis-related genes,such as TRAF2, CHOP, were activated by MCs. Taking into consideration the mechanisms of apotosis caused by microcystins are different with exposure to the different concentration and time, so, we presumed the apoptotic pathway could be ASKl-JNK and CHOP. Glueose-regulated protein 78 is the key gene during the ERS, which could repress and activate the related genes to control the occurrence of apoptosis. In this study, grass carp from Poyang Lake and hepatocyte cultured in vitro will be exposed to microcystin-LR,one of the most hepatatoxin of microcystins, to build experimental model. The pathways and mechanisms of apoptosis mediated role of ERS will be studied by using flow cytometry, western blot and so on. Futhermore, in order to study the roles of regulating ERS-mediated apoptosis threatened by microcystin-LR and its signaling pathway, overexpression and short interfering RNA will be used to interfere the expression of GRP78. Meanwhile, high-throughput sequencing will be used to screen the downstream genes of GRP78 so as to understand its regulatory pathways deeply. This work can provide not only better understanding of the hepatotoxic mechanisms and prevention of microcystin-LR, but also more scientific basis for monitoring of MC-LR of water in Poyang Lake and security evaluation of fishery production.

鄱阳湖是我国最大的淡水湖，日益面临着富营养化的威胁。水体富营养化可产生微囊藻毒素(MCs)，其高度的肝毒性已引起科学界广泛关注。诱导细胞凋亡是MCs重要毒性机制之一，但由内质网应激(ERS)介导的凋亡调控机制尚不清楚。我们前期研究初步表明MCs可致鱼类细胞凋亡并激活ERS相关基因TRAF2和CHOP等，我们推测两条ERS信号通路ASKl-JNK和CHOP可能参与了MCs诱导草鱼肝细胞凋亡。基于以上原因，本项目拟以鄱阳湖区草鱼作为研究对象，采用流式细胞术、免疫印迹等方法进一步研究ERS介导MCs诱导细胞凋亡的具体途径，揭示ERS介导凋亡的作用机制；同时采用过表达、RNA干扰和高通量测序技术研究ERS的信号分子GRP78对MCs诱导细胞凋亡的调控作用，揭示其调控机制。该项目的完成将诠释MCs肝毒性的新机制，为MCs的防治提供参考资料，亦为鄱阳湖水体污染的生物监测及渔业生产安全评估提供可靠依据。

水体富营养化可产生微囊藻毒素(MCs)，其高度的肝毒性已引起科学界广泛关注。本项目以草鱼作为研究对象，采用电镜研究了不同剂量MC-LR对草鱼肝细胞内质网的影响，发现随着暴露剂量的增加，内质网应激更明显；通过TUNEL发现MC-LR可诱导草鱼肝细胞产生凋亡，并且也具有剂量依赖效应。从转录组角度研究了MC-LR对草鱼肝脏的影响，结合生物信息学分析了其响应MC-LR胁迫的转录组水平变化，筛选到大量与内质网应激、细胞凋亡等相关的差异表达基因和信号通路，并重点对内质网应激相关的ASKl-JNK和CHOP两条通路以及凋亡通路上的基因（Caspase-8-like、IRE1-Like、Kinase-Pim1、TP53INP1、 Rab-1A和AIF）进行了克隆，并制备了相应的抗体，从蛋白水平上检测了不同剂量MC-LR诱导其表达变化。同时也对内质网应激相关通路上的GRP78、GRP94、ATF4、ATF6和XBP1等进行转录水平分析，发现这些基因可被诱导表达，尤其是GRP78在不同剂量MC-LR处理后均可被显著诱导，这些结果也表明MC-LR可对内质网应激产生影响并激活了这些通路。此外，在对转录组数据进行分析的同时，发现大量与免疫相关的差异表达基因和信号通路也发生了变化，尤其是补体相关的基因和信号通路变化更为显著。因此，还对草鱼免疫器官脾脏、头肾和肠道以及B淋巴细胞发育相关和炎症相关的基因进行了分析，研究结果表明MC-LR可导致草鱼免疫器官一系列的病理变化，并具有时间和剂量依赖效应。另外，MC-LR还可抑制B淋巴细胞分裂相关基因的表达，而低剂量MC-LR可促进促炎性细胞因子IL-1β、IL-8和TNF-α等的表达。此外，还研究了MC-LR对草鱼抗氧化防御系统的影响，对草鱼抗氧化酶和抗氧化相关的基因的检测分析，发现MC-LR对草鱼肝脏抗氧化系统产生了明显的胁迫效应。对MC-LR毒性的研究，不但具有重要的理论意义，而且还可为水体中MC-LR污染的生物监测及渔业生产安全评估提供可靠依据。