| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所碩士班 === 93 === Living cells are continually challenged by various acute and chronic stresses. Heat shock proteins (HSPs) are believed to be the most ancient defense system and efficient cytoprotective mechanism in all living organisms. Increased induction of HSPs occurs after heat shock, infection, heavy metals, and environmental stresses. These proteins act as molecular chaperones by helping in refolding of misfolded proteins and assisting in their elimination if they become irreversibly damaged. Growing evidence has shown that environmental and pathological stresses induce HSPs, especially the inducible form of HSP70, and induction of HSPs protect cell from subsequent lethal stresses. However, a recent study shows that chronic exposure of chick embryos to electromagnetic field decreases HSP70 induction and results in decline of cytoprotection. Therefore, it is important to know what the real effect of chronic heat shock is. We have previously reported that single heat shock pretreatment of cultured cells induces HSP70 to against TNFα induced apoptosis. In this study, we designed a repetitive heat-shocked cell model of hepatocyte cell line, clone 9, and detected HSP70 and Heat shock factor-1 levels by Western blot analysis and RT-PCR. Apoptosis was evaluated by in situ TUNEL stain and Flow cytometry. Our findings are : (1) Single and double heat shock pretreatment of clone 9 cells induced protection against TNFα- induced apoptosis, but three times of heat shock pretreatment did not. (2) There are no difference between single and repetitive heat shock pretreatment of clone 9 cells in HSP70 content of total cellular extract as well as cytoplasmic or nuclear extract. (3)Phosphorylation of Heat shock factor-1 decreased accordance with the frequency of heat shock pretreatment. (4) Single and double heat shock pretreatment of clone 9 cells induced larger amount of hsp70 mRNA, but triple heat shock pretreatment induced significantly few hsp70 mRNA. We suggest that decline of Heat shock factor-1 activation followed by decreased induction of newly synthesized Hsp70 might lead to the disappearance of cytoprotection after more than three times of heat shock treatment.
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