The Relationship Of Heat Shock Protein 90 And The Cylin B1 In Oxidative Stress

BackgroudOxidative stress refers to various harmful stimulus from external and internal environment of the body, the body produce reactive oxygen species (ROS clusters, ROS) and active reactive nitrogen (n species, cluster RNS), cause physiological and pathological reaction. Because they can directly or indirectly oxidation or damage proteins, lipids and DNA, induce protein denaturation, lipid peroxidation and genetic mutations. Oxidative stress is a "double-edged sword":on one hand oxidatie stress is one of the most basic protection mechanism of the body, regulate transcription factor by regulating signaling pathways of the body; It takes part in inflammation, immune, cell proliferation and cell regeneration, repair process etc in the body; On the other hand oxidatie stress exceed body antioxidant system to eliminate ability,it can lead to lots of savings of ROS,and then caused the damage to cells and tissues, and participate in cardiovascular disease, organ fibrosis, wound healing delay, diabetes, neurodegenerative and aging and tumors process at the same time. In view of the oxidative stress two-way function, there has been related reported that it has been used to regulate the REDOX state, initially applied for cardiovascular preconditioning protection and tumor treatment through promoting and antioxidant.but not so good for a variety of other disease's treatment,these results suggest the complexity of oxidative stress, is not a simple ROS state regulation. Therefore, exploring oxidatie stress reaction and damage caused by the body protective signaling pathways are important to clarify specific molecular mechanism under oxidatie stress pathways, disease prevention and treatment of drug compatibility. under special environmentQuality control system and is closely related to cell cycle regulation, and Hsp90 as an important partner quality-control system protein molecules is vital to maintain the normal cell function and survival under oxidatie stress. Oxidative stress causees the cell cycle blocked, wether Hsp90 participation in the adjustment of cell cycle protein, how is the interaction? The clalifying of the mechanism is helpful for the research of mechanisms of the disease related to oxidatie stress and prevention, and new drug development.Objectives1. To study whether oxidatie stress treatment influence the HepG2 cell cycle;2.To research the expression of HepG2 cells Cyclin B1 and Hsp90a, their positioning and interaction under oxidatie stress condition;3. The GFP-Hsp90aE47A was mediated by slow virus into HepG2 cells, thus competitive inhibiting cells Hsp90a ATPase activity;4. In the previous step on the basis of detection of Hsp90 and Cyclin B1 changes and analysis the interaction between Hsp90 and the Cyclin B1, and in order to further investigate mechanism of cell cycle arrest and the heat shock proteins in cells under oxidative stress in the molecular mechanism of protection. Methods1. Group of oxidative stress in HepG2 cells:The cells were divided into control group of control, group of oxidative stress 2h,4h group,6h group,8h group,12h group,24h group.Oxidative stress factors:500μmol/L H2O2, were to stimulate the appropriate time.2.Flow cytometry is used to detect cell cycle of cells;3. western-blotting is used to detect content of Protein carbonyl, Hsp90α,Cyclin B1,ubiquitin,ubiquitinated Cyclin B1 and so on;4. Fluorescence microscopy is used to observe GFP-Hsp90αE47A;5. Quantitative Real-time PCR is used to detect Cyclin B1mRNA level.6. Immunofluorescence and CO-IP to detect stress on intracellular synthesis of Hsp90 and the Cyclin B1, intracellular co-localization and cell interactionsResults1. western-blotting detection of oxidative stress on the expression of Protein carbonyl results:Protein carbonyl status was presented by anti-DNP immunoblot, and became severer with the prolongation of H2O2 treatment;2. Flow cytometry detection of oxidative stress HepG2 cells cycle results: oxidative stress HepG2 24h presented obvious G2/M phase arrest;3.western-blotting detection of oxidative stress on the expression of Hsp90αand Cyclin B1 results:oxidative stress lead to increased expression of intracellular Hsp90a at 6h and 8h,12h,24h decreased to normal; Cyclin B1 increased, ubiquitinated Cyclin B1 increased first, then decreased;4.Fluorescence microscope and Lentiviral transfection results of western-blotting after transfection, the identification results:Hsp90αE47A/psin-GFP gene transfected cells under fluorescence microscope, green fluorescence was observed; Hsp90aE47A gene transfected cells, and untransfected control cells compared with obvious exogenous expression of GFP-Hsp90aE47A.5. RT-PCR detection of oxidative stress HepG2 and Hsp90a E47A mutant cells on the expression of Cyclin B1mRNA results:oxidative stress HepG2 and Hsp90a E47A mutant cells didn't present increased level of Cyclin B1 mRNA; western-blotting detection of oxidative stress on the expression of Cyclin B1 of results:oxidative stress lead to increased expression of intracellular Cyclin B1; Hsp90a E47A mutant cells presented obvious increased level of Cyclin B1, either under normal status or after 6 hours H2O2 treatment;Flow cytometry detection of oxidative stress HepG2 and Hsp90a E47A mutant cells cycle results:oxidative stress HepG2 and Hsp90a E47A mutant cells presented obvious G2/M phase arrest.6. Immunofluorescence staining cells were Hsp90 and the Cyclin B1 localization result:Immunofluoresence images indicated, Cyclin B1 accumulated exactly at the area where presented low level of Hsp90a;CO-IP is used to detect interaction of Hsp90 and the Cyclin B1:with oxidative stress process Hsp90 and the Cyclin B1 combined more,group 24 is higher than control;7. western-blotting detection of oxidative stress on the expression ubiquitin and ubiquitinated Cyclin B1 results:oxidative stress lead to decreased expression of ubiquitin, increased expression of ubiquitinated Cyclin B1.Conclusions1. H2O2 effect on HepG2 cells at different times, cells present different degrees of damage.In 24h, the effect of longer intracellular the more Protein carbonyl.2. Oxidative stress can affect cellular expression of Hsp90a and the Cyclin B1 and change its cellular distribution and interaction between the two, suggesting that in this process, Hsp90α,together with oxidative stress, plays a complex role on Cyclin B1 accumulation.3. Successfully make GFP-Hsp90αE47A stably expressed by lentiviral gene in HepG2 cells, in order to build this new cell line to establish a basis for subsequent experiments.