中文摘要：

目的观察人肝癌细胞氧化应激模型中细胞内Hsp90α和20S蛋白酶体的表达的变化以及Hsp90α表达下调后对20S蛋白酶体功能的影响;明确热休克蛋白90α对20s蛋白酶体功能的影响。方法以500μMH2O2建立氧化应激HepG2细胞模型;通过ROS探针法观察细胞在不同氧化应激条件下ROS的分布和含量,通过MTT比色法观察氧化应激对细胞存活的影响;以免疫印迹方法检测氧化应激对细胞内Hsp90α及20S蛋白酶体的合成的影响;以pSilencer2.1-U6neo载体,用电穿孔法建立稳定的Hsp90α抑制表达细胞株,通过检测糜蛋白酶活性观察氧化应激和降低Hsp90α表达对蛋白酶体活性的影响。结果氧化应激后细胞内ROS的含量增多,且分布从胞浆向胞核转移;随着作用时间延长,H2O2对HepG2细胞增殖的抑制程度增强;氧化应激导致胞内Hsp90α表达量先增加后降低,20S蛋白酶体表达量明显降低;siRNA干扰组蛋白酶体活性显著下降。结论氧化应激可影响细胞内Hsp90α和20S蛋白酶体的表达并改变其细胞内分布;氧化应激及降低Hsp90α的表达均可影响蛋白酶体的活性;Hsp90α对蛋白酶体功能维持起保护性的作用。

英文摘要：

Objective To investigate the change of Hsp90α and the 20S proteasome in HepG2 cells under the conditions of oxidative stress.To detect the changes of proteasome activity after lower the expression of Hsp90α,and to further explore the molecular mechanism of the protective function of heat shock protein 90α under oxidative stress.Methods Oxidative stress model in HepG2 cells was established by 500 μmol/L H2O2,using ROS probe to observe the distribution and content of ROS in cells under different conditions,and using MTT to observe cell survival.The intracellular synthesis of Hsp90α and 20S proteasome was etected by Western-blotting.The recombinant plasmid pSilencer Hsp90 was introduced into HepG2 cells by electroporation,to detecting the proteasome chymotrypsin activity after lowering the expression of Hsp90α.Results ROS significantly increased in HepG2 cells after oxidative stress and distribution of ROS transfer from cytoplasm to the nucleus.Under 500 μM H2O2 concentration,the inhibition of cell proliferation was time dependent.With the time prolongation,the inhibition gradually increased.Oxidative stress led to first increased and then decreased expression of intracellular Hsp90α,but the expression of 20S proteasome significantly reduced;after stable siRNA interference,proteasome activity had a more significant decline.Conclusions Oxidative stress can affect the expression of Hsp90α and 20S proteasome and the changes of distribution and interaction between the two proteins,suggesting that in this process,Hsp90α plays a protective role,reduces the direct damage caused by stress,and maintains the stability of proteasome function.