热休克蛋白90在预热适应成纤维细胞中作用

Effect of Hsp90 on heat preconditioning NIH-3T3 fibroblast

目的建立小鼠成纤维细胞株NIHF-3T3应激适应细胞模型,探讨热休克蛋白90(HSP90)在应激适应中的作用及机制。方法通过预热适应(42℃,20min)建立应激适应细胞模型,并通过再次热应激时(44℃,40min)细胞膜损伤指标、DNA损伤指标、细胞形态学改变综合评价适应效果。以免疫细胞化学检测应激适应对细胞内HSP90合成和细胞内定位的影响。结果结合预适应后再次热应激所致的损害情况,初步确定预适应后6h为最佳应激保护时间。再次热应激时,细胞膜损伤指标、DNA损伤指标、细胞形态改变均表明预适应后预处理(42℃,20min)6h后,再次热应激时,培养液中乳酸脱氢酶(LDH)漏出变化率、细胞DNA受损较直接热应激组为轻,细胞损伤状态减轻。热应激40min后细胞内HSP90含量均呈现下降趋势,并伴有细胞内的重新分布。结论通过对NIH-3T3细胞进行预适应处理,通过观测细胞膜损伤、DNA损伤、细胞形态变化情况,确定应激保护的时间点,建立了细胞应激适应模型,初步确认HSP90在该模型中的保护作用。

Objective To establish stress adaptaion model of mouse fibroblast cell line NIH-3T3, and to provide a group of parallel object for stress adaptation research. To explore the function and mechanism of HSP90 in stress adaptation. Methotis A stress-adapted cell model was established by thermal preconditioning （42℃,20 min）. Adaptation situation was evaluated by the membrane injury （To measure the activity of lactate dehydrogenase in supernatant by automatic biochemistry analyzer）, damage of DNA （To measure the PI content pentrating into DNA by FCM） and th changes of cell morphology. HSP90 content and location were deteced by immunocytochemistry. Results Combining the membrane injury and HSP90 synthesis immediately after heat stress, 6h after thermal preconditioning was confirmed the ideal stress protection time. When cells faced heat sress 6 h after thermal preconditiong, the membrane injury, damage of DNA and the changes of cell morphology were alleviated compared with control group which was heated without preconditioning. Cellular HSP90 contets decreased immediately after heat stress （44℃,40 min）. HSP90 located in cytoplasm under physiological situation and were recruited into nucleolus under stress condiion. Conclusion Thermal preconditioning may promote the relocation of HSP90 under stress condition. Cellular stress adaptation was established by NIH-3T3 cell line thermal preconditioning. Stress prodection of HSP90 was confirmed in this model. The effect of high-level HSP90 on cell may show protection under severe cirumstance, through HSP90 relocation and protectiong the vital proteins in stress signal transduction pathway.