| BackgroudThough stress injury has been explored thoroughly, the mechanisms of stress and adaptation still need to be elucidated during the complex interaction of environment to organism, especially at the initial point. Adaptation is a harmonized biological adjusted procedure involved multiple system, the foundation of which is a cooperative action of signal cascade transduction network and multi-genes. During the investigation of environmental adaptation, the molecular mechanism remains unclear, though the physiological and biochemical characters are well understood, so it is necessary to do further research on known stress-related molecular and screen for unknown ones.Objectives1. Establish stress adaptation model of mouse fibroblast cell line NIH-3T3, and provide a group of parallel object for stress adaptation research.2. Explore the function and mechanism of HSP90 in stress adaptation by construct hsp90 highly expressing cell lines.3. Analyze the effect of stress and adaptation on protein synthesis, and screen for stress adaptation related proteins.Methods1. A stress-adapted cell model was established by thermal preconditioning(42℃, 20min) ,a nd adaptation situation was evaluated by the membraneinjury (measured the activity of lactate dehydrogenase in supernatant byautomatic biochemistry analyzer) , damage of DNA (measured the PI contentpenetrating into DNA by FCM) and the changes of cell morphology.2.HSP90 content and location were detected by Western blot and immunocytochemistry.3. The recombined plasimid pSmycHSP, which contains the full length DNA coding for human hsp90 β ,was introduced into mouse fibroblast cell line NIH-3T3 by electroporation after being subcloned purified andidentified by limited enzyme digestion. Screened by G418, the positive clones were selected and identified by immunofluorescence and Western-blotting. The proliferationof new cell lines were measured by MTT method.Contrasted with NIH-3T3 cells transfected with empty plasmid, hyperthermia(44,40min)was used to simulate oxidative stress. The activity of lactate dehydrogenase (LDH) in supernatant, damage of DNA and HSP90 substrate protein binding qualitity were detected by automatic biochemistry analyzer, flow cytometer separately to analyze the effect of high-level HSP90 on cell membrane, DNA injuries under stress condition.4. HSP90 binding abilities of substrate protein HSP70 Raf-lwere observed by HSP90 coimmunoprecipitation.The relationship between stress injury/adaptation and change of binding state was analyzed.5. Total cytolytes were separated by 2-DE, analyzed by PDQUEST software, and the selected diferential expressing spots were detected by MOLDI-TOF. The effect of stress and adaptation on protein synthesis was studied, and stress adaptation related spots were identified by PMF.Results1. Combining the membrane injury and HSP90 synthesis immediately after heat stress, confirmed 6h after thermal preconditioning was the ideal stress protection time. When cells faced heat stress 6h after thermal preconditioning, the membrane injury (measured the change percent of lactate dehydrogenase activity in supernatant by automatic biochemistry analyzer, 15.4?.6 v.s. 41.2?.1, t test, <0.01) ), damage of DNA (measured the PI content penetrating into DNA by FCM, 15.1% v.s. 26.3%, jc2test, /K0.01) and the changes of cell morphology were alleviated compared with control group which is heated without preconditioning.2.Cellular HSP90 contents decreased immediately after heat stress(44, 40min). HSP90 located in cytoplasm under physiological situation and were recruited into nucleolus under stress condition. Thermal preconditioning might promote the relocation of HSP90 under stress condition.3. The increased level of HSP90 in transfected cell line was shown by immunofluorescence and Westen-blotting. Compared with control, the leakage of LDH (U/L) (1.3?.6 v.s. 9.3?.7, t test ,p<0.001) and damageof DNA (17.2% V.S.24.2%, x2 test, /?<0.01)were relieved under 44,40mino4. The change of...
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