RIP1/3介导灿烂弧菌感染过程中刺参不同细胞死亡方式转换的分子机制

RIP1/3作为细胞生存和死亡的重要交叉点和转换器，在细胞存活、凋亡与坏死等过程中发挥关键性作用。申请人前期研究发现灿烂弧菌诱导下刺参4种细胞死亡方式与RIP1/3的表达显著相关。但对于该病原诱导的细胞死亡方式之间的转换是否受RIP1/3调控？调控方式是什么以及改变细胞死亡方式转换对腐皮综合症发展如何影响等关键科学问题尚不明晰。为此，本项目拟通过免疫荧光、电镜等技术，明确刺参不同发病阶段与细胞死亡方式的相关性；通过激活剂、RNAi等技术，解析RIP1/3介导的刺参体腔细胞死亡方式之间相互转换的分子机制；通过定点突变及磷酸化western blot检测，阐明RIP3磷酸化MLKL导致刺参体腔细胞坏死的分子途径；通过RIP1/3抑制剂处理，揭示细胞死亡方式改变对刺参腐皮综合症发生发展的影响。研究成果将为认识和定向防治刺参腐皮综合症提供新思路。

As an important cross point and switch between cell survival and death, receptor-interacting protein 1/3 (RIP1/3) plays a key role in regulation cell survival, apoptosis, pyroptosis and necrosis. Our previous studies showed that the four types of cell death were detected in Vibrio splendidus challenged Apostichopus japonicus, and also significantly related to the expression of RIP1/3. However, it remains unclear whether RIP1/3 directly regulates the different cell death manners, how does it do that, and the connection between cell death switch and the occurrence of SUS. In this project, we intend to illustrate the molecular mechanism of cell death under V. splendidus exposure. Through immunofluorescence, scanning electron microscope and other techniques, we will demonstrate the relationship between different stages of SUS disease and form of cell death of sea cucumber. Through activator and RNA interference techniques, we will analysis of the molecular mechanism of RIP1/3-mediated the switching of different cell death. Through site-specific mutagenesis and analysis phosphorylation level, we will certify the molecular mechanism of cell death regulation of RIP3 is determined by the specific sites phosphorylation of MLKL. Finally, through inhibitor treatment, we will to reveal the effect of changing the form of cell death during SUS disease of sea cucumber. In all, the results of this project will provide new ideas for understanding and directing control of SUS disease.