氧化应激对Hsp90α、ARF1细胞内定位和相互作用的影响

The Effects of Oxidative Stress on Intracellular Localization and Interaction of Hsp90 and ARF1

目的:探讨氧化应激对热休克蛋白90α(Hsp90α)与ADP-核糖基化因子1(ARF1)细胞内定位、相互作用的影响。方法:应用500μM H2O2处理HepG2细胞,建立氧化应激模型,MTT比色法检测细胞活力,Western blotting检测Hsp90α和ARF1水平,细胞免疫荧光法、免疫共沉淀检测上述蛋白在氧化应激下的分布、共定位变化和相互作用。结果:MTT比色法结果提示,随氧化应激时间延长,细胞存活力降低;Western blotting结果显示,氧化应激可提高胞内Hsp90α和ARF1蛋白水平;免疫共沉淀结果显示,随氧化应激作用时间延长,Hsp90α与ARF1相互结合增多;细胞免疫荧光结果显示,随氧化应激作用时间延长,Hsp90α与ARF1荧光强度增强,并趋于沿胞膜分布。结论:提示氧化应激影响Hsp90α和ARF1的水平、胞内分布及相互作用。

Objective： To investigate the effect of oxidative stress on the translocation and relationship of heat shock protein 90α （Hsp90α） and ADP-ribosylation Factor 1 （ARF1）. Methods： 500μM H2O2 were used to induce oxidative stress in HepG2 cells. MTT（3- （4,5）-dimethylthiahiazo（-z-yl）-3,5-di-phenytetrazoliumromide） assay. Western blotting, immunofluoresence and co-immunoprecipitation were carried out to identify cell viability, protein level, protein translocation and interaction. Results： MTT colorimetry showed that with the prolongation of oxidative stress treatment, cell viability decreased. The effect of Western blotting demonstrated that oxidative stress increased the levels of Hsp90α and ARF1 in HepG2 cells, increased ARFI and Hsp90α colocalization. The co-immunoprecipitation results showed that with the extended of oxidative stress, Hsp90α and ARF1 combined increased. Asto immunofluoresence, it is also observed that treatment, when H2O2 induced oxidative stress prolonged, fluorescence intensity of the two proteins had increased, the proteins tended to range near the cell membrane. Conclusion： Oxidative stress affected the expression level of Hsp90α and ARF1 in HepG2 cells, the intracellular distribution and interaction were also involved.