葡萄糖调节蛋白94及其互作蛋白调控内质网应激诱导的猪肝细胞凋亡机制

Glucose-regulated protein 94 (GRP94) is a marker protein of endoplasmic reticulum stress, which is essential for the stability of endoplasmic reticulum in the cell stress state. Studies have shown that endoplasmic reticulum stress can induce apoptosis, but it is not clear how GRP94 and its interacting protein participate in the activation of apoptosis. In order to solve the scientific problem, the project is intended to carry out four aspects of research one by one: (1) The expression of GRP94 and the activation of main apoptotic pathway in the liver of endoplasmic reticulum stress pigs will be analyzed by intravital injection and dose effect method, and a porcine endoplasmic reticulum stress model will be established; (2) On the basis of the porcine endoplasmic reticulum stress model, the relationship between the low expression of GRP94 and the apoptosis, apoptosis signaling pathway of porcine liver will be proven up by in vivo inhibitor injection; (3) By GST pull-down, co-immunoprecipitation and other methods, one GRP94 interacting protein X with significant change in apoptosis signaling pathway will be obtained through screening and validating from animal samples of two previous studies; (4) Primary cultured porcine hepatocytes will be prepared to explore whether the expression of knockdown GRP94 interacting protein X affects the apoptotic signaling pathway of hepatocytes under endoplasmic reticulum stress, in order to reveal the molecular mechanism of GRP94 interacting protein regulating cell apoptosis. The expected results will provide a new idea for the regulation of the livestock production conditions which can induce endoplasmic reticulum stress and the clinical prevention and treatment of liver disease related to endoplasmic reticulum stress.

葡萄糖调节蛋白94（GRP94）是内质网应激（ERS）的标志蛋白，在细胞应激下对稳定内质网内环境至关重要。已有报道，ERS可诱导细胞凋亡，但GRP94如何参与细胞凋亡的活化还不清楚。本项目针对此科学问题，拟递进地开展四方面研究：①通过活体注射法，分析ERS猪肝脏GRP94表达和主要凋亡通路活化情况，建立猪ERS模型；②在猪ERS模型基础上，采用化学试剂抑制法，探明猪肝脏GRP94低表达与其细胞凋亡、凋亡信号通路的关系；③利用GST pull-down结合、免疫共沉淀等手段，从前述研究动物样本筛选、验证获得细胞凋亡信号通路中变化显著的一个GRP94互作蛋白X；④制备原代培养的猪肝细胞，探究敲低GRP94互作蛋白X的表达是否在内质网应激条件下对肝细胞凋亡信号通路产生影响，旨在揭示GRP94调控细胞凋亡的分子机制。结果可为畜牧上引起ERS的生产条件调控、临床上与ERS相关的肝病防治提供新的思路。

针对GRP94如何调控细胞凋亡的科学问题，本项目递进地开展了四方面研究：①通过活体注射适宜浓度的衣霉素（0.1 mg/kg体重），成功建立了仔猪内质网应激模型，初步明确内质网应激条件下GRP94表达与猪肝细胞凋亡的关系；②利用慢病毒介导的shRNA转染永生化猪肝星状细胞，建立了GRP94低表达的内质网应激细胞模型，检测细胞增殖和凋亡状况、细胞内质网标志蛋白及其相关信号通路、IGF-1表达和泛素修饰的变化，明确了内质网应激条件下GRP94影响肝细胞凋亡的机理；③利用蛋白免疫印迹、免疫共沉淀等手段，富集与质谱鉴定正常培养条件下或内质网应激条件下猪肝星状细胞中GRP94互作蛋白，并对鉴定到的内质网应激条件下与GRP94有互作关系的76个蛋白进行GO、KEGG和互作关系网络分析，筛选、验证到细胞凋亡信号通路中变化显著的GRP94互作蛋白——波形蛋白；④利用慢病毒介导的shRNA转染永生化猪肝星状细胞，建立了波形蛋白低表达的内质网应激细胞模型，测定了细胞增殖和凋亡状况、细胞内质网标志蛋白及其相关信号通路、脂代谢相关酶的变化，明确了内质网应激条件下波形蛋白表达对肝细胞凋亡和脂肪代谢的影响，首次揭示了GRP94调控通过波形蛋白调控细胞凋亡的分子机制。结果可为畜牧上引起ERS的生产条件调控、临床上与ERS相关的肝病防治提供新的思路。