GRP94对高温应激诱导小黄鱼肝脏细胞凋亡及其信号分子IRE1α的调控作用研究

高温应激造成养殖鱼类肝脏损伤，给水产养殖带来巨大影响，备受人们关注。前期研究发现高温应激导致小黄鱼肝脏细胞凋亡、内质网应激标志蛋白GRP94（葡萄糖调节蛋白94）基因和细胞凋亡关键信号分子IRE1α（肌醇需求激酶1α）基因均显著上调表达。推测高温应激导致内质网应激，进而激发IRE1α介导的细胞凋亡的发生，但是IRE1α对高温应激诱导细胞凋亡的响应机制以及GRP94在此过程中的作用机理尚不清楚。本项目拟开展小黄鱼的体内实验和小黄鱼肝细胞的体外实验，采用高通量测序、实时荧光定量PCR、流式细胞术、免疫组化、蛋白免疫印迹、抑制和过表达等手段，解析GRP94和IRE1α对高温应激诱导细胞凋亡的响应机制、GRP94对IRE1α及其介导的细胞凋亡的调控作用。本项目将进一步揭示鱼类肝脏响应高温应激的分子调控机制，为从提高肝脏内源性保护角度防治高温应激诱导的肝病提供科学依据。

Heat stress, which causes liver damage in farmed fish, has an enormous impact on aquaculture and has thus attracted widespread attention. Preliminary studies found that heat stress caused liver apoptosis, and both the genes of endoplasmic reticulum stress marker protein GRP94 (Glucose regulatory protein 94) and the key apoptosis signal molecule IRE1α (Inositol-requiring enzyme 1α) were significantly up-regulated in the small yellow croaker, Larimichthys polyactis. We speculated that heat stress leads to endoplasmic reticulum stress, which in turn stimulates the occurrence of IRE1α-mediated apoptosis. However, the response mechanism of IRE1α to heat stress and the mechanism underlying GRP94 action still remain unclear. This project intends to carry out both in vivo and in vitro experiments to explore the response mechanisms of GRP94 and IRE1α to heat stress, and the regulatory effect of GRP94 on IRE1α and apoptosis, using the high-throughput sequencing, RT-qPCR, flow cytometry, immunohistochemistry, Western blotting, as well as the inhibition and overexpression of specific genes. This project will work toward identifying the molecular regulation mechanism that is active in the fish liver in response to heat stress. Furthermore, a scientific basis for the prevention and treatment of heat stress-induced liver diseases will be provided from the perspective of improving liver endogenous protection.