多次熱休克處置對腫瘤壞死因子α誘發肝細胞凋亡之效應探討

Effect of repetitive heat shock stimulation on TNFα- induced apoptosis in clone 9 cells

高久晴(Chiu-Ching Kao)

指導教授 ： 楊瑞成

高雄醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

細胞不斷地挑戰來自周遭環境中急性或慢性的窘迫刺激，而熱休克蛋白家族組成了生物細胞內最古老的防禦系統。即當細胞受到熱、重金屬、病毒、或藥物等外來因子的刺激後，會因應這些刺激而誘發產生熱休克蛋白，這些熱休克蛋白具有保護的功能，幫助新合成或受破壞蛋白質的折疊與復原，使其具備應有的正確結構和功能，或者將無法修復之蛋白質進行分解。其中，熱休克蛋白家族中以熱休克蛋白70，最具有誘發性。但在另外的文獻指出，給予雞胚胎長時間的電磁波刺激，卻導致熱休克蛋白70表現量減少，隨後再讓雞胚胎缺氧，結果雞胚胎死亡率明顯上升，顯示長時間的電磁波刺激，已使熱休克蛋白70喪失保護細胞的能力。到底多次誘發刺激對於細胞的效應為何，其保護機制是否有所變化是急待釐清的課題。在之前本實驗室的研究結果顯示，予以體外肝細胞單次加熱處理來誘發熱休克蛋白70後，呈現熱休克蛋白70大量表現，再加入腫瘤壞死因子（TNFα）誘發細胞凋亡，發現細胞凋亡的現象明顯減少。本實驗設計乃是將細胞經過多次加熱處理後，再加入腫瘤壞死因子誘發細胞凋亡，以流體細胞儀及TUNEL染色法偵測細胞凋亡的情形，再以西方點墨法和RT-PCR測量熱休克蛋白70的表現及熱休克轉錄因子-1活化狀況。結果呈現三次加熱前處理:(1) 細胞凋亡現象無明顯減少 (2) 細胞內熱休克蛋白70含量維持與單次加熱相同。 (3)熱休克轉錄因子-1活化程度依加熱次數遞減。(4) 多次加熱處理後，熱休克蛋白70 mRNA表現量減少。推測細胞經過多次加熱處理後，已無明顯保護細胞的作用；其原因可能是表現在細胞中的熱休克蛋白70，經負回饋路徑和熱休克轉錄因子結合，導致降低熱休克轉錄因子-1的活化及新的熱休克蛋白70合成減少所致。

關鍵字

多次熱休克

並列摘要

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.

並列關鍵字

repetitive heat shock

參考文獻

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