海参自溶过程中钙网蛋白介导内质网应激相关凋亡的信号通路研究

Sea cucumber with the strong ability of autolysis introduces a serious problems with respect to its storage, preservation and processing. Previously results have showed that, carlreticulin (CRT) upregulated during sea cucumber autolysis and accompanied by apoptosis. And over-expression of CRT could trigger endoplasmic reticulum stress (ERS)-related apoptosis. However, the issues are not clear that whether ERS-related sea cucumber apoptosis triggered sea cucumber autolysis, and how the signaling pathway of CRT, as the key factor, induces ERS-related apoptosis. This project intends to figure out the signaling pathway of apoptosis during sea cucumber autolysis. Firstly, based on the models of UV-induced sea cucumber autolysis and tunicamycin (TM)-induced ERS, cell apoptosis and necrosis will be assessed. ER specific resident fluorochrome Dapoxyl will be used to observe ER morphological changes. RT-PCR and/or Western blotting will be used to detect expression of ERS molecules and apoptosis-related molecules. It will suggest that ERS induces sea cucumber apoptosis and then triggers sea cucumber autolysis. Secondly, in order to investigate whether CRT is the key factor to induce ERS-related apoptosis during sea cucumber autolysis, specific interference agents will be used to interfere with CRT expression. Finally, the signaling pathway of CRT-mediated ERS apoptosis during sea cucumber autolysis will be investigated basing on the model of UV-induced sea cucumber autolysis with over-expression of CRT. Thus, the results of this research would lay the theoretical foundation for revealing the mechanism of sea cucumber autolysis.

海参极强的自溶能力给其贮藏、保鲜和加工带来诸多难题。研究发现，海参自溶时钙网蛋白表达上调，且出现细胞凋亡特征。钙网蛋白过表达可触发内质网应激及相关凋亡。 然而，对于内质网应激是否引起海参自溶，钙网蛋白作为应激关键分子如何介导凋亡等问题，目前仍未明确。 本项目拟从凋亡信号传导的角度研究海参自溶现象。首先，在紫外诱导海参自溶和内质网应激诱导剂作用的海参细胞模型上，采用细胞损伤检测、RT-PCR和/或Western blotting检测确定内质网应激相关凋亡引起海参自溶；其次，采用特异性干扰剂调节钙网蛋白上调或抑制表达，确定海参自溶时钙网蛋白是引起内质网应激相关凋亡的关键分子；最后， 在钙网蛋白干预表达的紫外诱导海参自溶细胞模型上， 进一步探明钙网蛋白介导内质网应激相关凋亡引起海参自溶的信号途径。 从而，为揭示海参自溶机理奠定理论基础。

刺参（Stichopus japonicus）极易发生自溶，给刺参加工贮运行业造成极大经济损失。刺参自溶过程中伴随着体腔细胞凋亡的现象，胞内Ca2+浓度发生改变，内质网也发生了形态损伤，只有揭示其与细胞凋亡相关性，明确刺参自溶机理，才能有效控制刺参自溶。.本项目以鲜活刺参为研究对象，从分子水平上明确刺参体腔细胞内Ca2+信号参与调控细胞凋亡和内质网应激（ERS）介导细胞凋亡的机制；从亚细胞（细胞器）水平上明确刺参体腔细胞内质网和线粒体损伤与细胞凋亡的相关性。.研究发现：..1.Ca2+信号参与调控体腔细胞凋亡的机制.刺参体腔细胞内Ca2+浓度随着静置时间的延长而逐渐升高，静置2h时达到最高值，随后下降；Ca2+-ATP酶活力、钙调神经磷酸酶活力和钙调蛋白分解酶活力均呈现先升高后降低的变化趋势；静置3h体腔细胞凋亡率达到最高值，随后降低。BAPTA-Na处理UVA组体腔细胞内Ca2+浓度、Ca2+-ATP酶、钙调神经磷酸酶、钙调蛋白分解酶以及细胞凋亡率均出现不同程度的下降，提示了Ca2+是参与调控刺参体腔细胞凋亡的起始信号。.2.内质网应激介导刺参体腔细胞凋亡的机制.内质网应激（ERS）介导体腔细胞凋亡相关因子CRT、Caspase-12、Caspase-9、 Caspase-3 和CHOP蛋白表达量均显著地增加，抗凋亡因子Bcl-2蛋白表达量则显著地减少，提示了在刺参自溶过程中体腔细胞的凋亡可能经历了内质网应激途径。.3.体腔细胞内质网应激与线粒体损伤.体腔细胞内质网荧光含量明显升高发生在2h，而线粒体荧光含量明显升高在1h时，揭示了刺参体腔细胞中线粒体结构损伤先于内质网结构损伤发生； Caspase-12与GRP78均在2h开始发生明显跃迁，而线粒体膜电位与线粒体复合体Ⅰ先后在1h与反应初始发生明显的下降，提示了线粒体也可能先于内质网发生功能损伤。.4.体腔细胞内质网与线粒体内钙离子分布.通过对内质网与线粒体内钙离子含量的测定，发现线粒体内钙离子浓度在实验初始阶段（0h）已发生明显变化，内质网中钙离子浓度明显改变发生在2h之后，提示了在刺参自溶过程中，线粒体先摄取细胞质中钙离子以维持自身平衡，内质网为维持细胞稳态平衡释放钙离子，导致自身结构功能受损。.综上所述，刺参自溶过程中，体腔细胞内Ca2+参与调控了细胞凋亡，其经历了内质网应激（ERS）途径。