HSP90高表达细胞株的建立及细胞应激反应的改变

Establishment of HSP90 overexpressing cell line and effects of HSP90 overexpressing on cell stress responses

目的 :建立稳定高表达热休克蛋白 90 (HSP90 )细胞株 ,研究HSP90高表达对细胞应激反应的影响。方法 :含人HSP90 β全长基因的重组质粒pSmycHSP经克隆、纯化、酶切鉴定后 ,用电穿孔法转染到小鼠成纤维细胞系NIH - 3T3细胞内。经G4 18筛选、克隆分离培养 ,用免疫荧光、免疫印迹鉴定阳性克隆。用高温作用 (44℃ ,2 0min、4 0min)模拟氧化应激环境 ,以转染空质粒的NIH - 3T3细胞为对照 ,全自动生化分析仪观测培养液中乳酸脱氢酶 (LDH)漏出活性变化、流式细胞仪测定DNA受损细胞数 ,分析细胞应激状态对细胞膜和DNA的损害及HSP90高表达对细胞应激反应的影响。结果 :转染pSmycHSP的NIH - 3T3细胞HSP90免疫荧光染色增强 ;与对照相比 ,4 4℃ ,2 0min时培养液中漏出的LDH无显著差别 ,DNA损害加重 ;4 4℃ ,4 0min时LDH漏出减少 ,DNA的损害减弱。结论 :已建立稳定高表达HSP90NIH - 3T3细胞株 ;HSP90高表达在不同的应激强度下对细胞的影响呈现复杂性 ,其应激保护作用与应激强度密切相关。

AIM: To establish a HSP90 highly expressing cell line and study the effect of high level of HSP90 on cell stress response. METHODS: The recombined plasimid pSmycHSP, which contains the full length DNA coding for human HSP90β, was introduced into mouse fibroblast cell line NIH-3T3 by electroporation after being subcloned, purified and identified by limited enzyme digestion. Screened by G418, the positive clones were selected and identified by immunofluorescence, immunocytochemistry and Western-blotting. NIH-3T3 cells transfected with empty plasmid served as control, hyperthermia(44 ℃, 20 min, 40 min)was used to simulate oxidative stress. The activity of lactate dehydrogenase (LDH) in supernatant and damage of DNA were detected by automatic biochemistry analyzer and flow cytometer separately to analyze the effect of high-level HSP90 on cell membrane and DNA injuries under stress condition. RESULTS: The rising level of HSP90 was shown by immunofluorescence, immunocytochemistry and Western-blotting in HSP90 overexpressing cell line. There was no difference in the leakage of LDH between HSP90 overexpressing cell line and control, but the damage of DNA was more severe at 44 ℃for 20 min in HSP90 overexpressing cell line than control. Compared with control, the above indices were relieved at 44 ℃ for 40 min in HSP90 overexpressing cell line. CONCLUSION: The NIH-3T3 derived cell line, which stably expressed high level of HSP90, was established. The effect of high-level HSP90 on cells is complex at different intensity of stress, and the protection may be shown at more severe stress circumstance.