熱休克前處理藉由避免淋巴球粒線體膜電位之去極化來保護過氧化氫所誘發之細胞凋亡

Heat shock pretreatment prevents H2O2-induced apoptosis in lymphocyte of rat by inhibiting the depolarization of mitochondrial membrane potential

李建志(Chien-Chih Lee)

指導教授 ： 楊瑞成

高雄醫學大學/醫學院/醫學研究所/碩士(2004年)

摘要

目前在重症加護病房最常見的病人致死原因即為敗血症及其所引發之多重器官衰竭。在近幾年的研究報告指出，無論是在敗血症之動物模式或是因敗血症所引發多重器官衰竭而死亡的病人，其顯示出在敗血症時會誘使血液中淋巴球經由細胞凋亡的方式而造成數目大量的減少。而在敗血症時，大量的淋巴球數目的減少會對病人個體的後天免疫系統造成嚴重的損害，使其無法對抗外來的病菌、病原體等。在我們實驗室先前的研究中顯示，大白鼠熱休克前處理可降低大白鼠敗血症時所引發死亡率，但其機制目前未明。在本研究，我們選用一個in vitro的動物模式，來偵測大白鼠在熱休克前處理後其對於過氧化氫所誘發淋巴球粒線體膜電位之去極化之保護作用。我們利用JC-1染劑來偵測粒線體膜電位之變化；西方點墨法來評估熱休克蛋白質72、Bcl-2、細胞色素c在細胞質及粒線體之含量且藉由流式細胞分析儀的Annexin-V及PI的雙染色法，及TUNEL染色法來觀察細胞凋亡的情形。我們的結果顯示：熱休克前處理可以保護淋巴球在過氧化氫的處理下，(1)免於細胞凋亡，(2)避免細胞色素c釋放至細胞質中，(3)穩固Bcl-2蛋白質在粒線體中的含量，(4)避免粒線體膜電位產生去極化現象。由以上的結果我們認為：大白鼠熱休克前處理可穩固粒線體中Bcl-2蛋白質之含量，避免粒線體膜電位之去極化及細胞色素c的流失來保護敗血症時細胞免於凋亡。

關鍵字

敗血症 ； 粒線體膜電位 ； 細胞凋亡 ； 熱休克蛋白72

並列摘要

Sepsis and the resultant multiple organ failure are the commonest cause of death in many intensive care units. Recent studies in animal models of sepsis as well as in patients who died of sepsis and multiple organ failure have shown that sepsis induces extensive loss of lymphocytes via apoptosis. Loss of lymphocytes in sepsis may be detrimental by impairing the ability of the immune system to combat pathogens. Our previous study showed that heat shock pretreatment reduced mortality in septic rats, but the mechanism is unclear. In the present study, we used an ex vivo cell model to detect the protective effect on H2O2-induced mitochondrial membrane potential depolarization of lymphocyte after heat shock pretreatment. The mitochondrial membrane potential was detected by JC-1 dye; and the Western blot analysis was used for evaluating the Hsp72, Bcl-2, and cytochrome c in the cytosome and mitochondria. Analysis of cell apoptosis was performed by Annexin-V and PI double staining flow cytometry and TUNEL stain. The results show that: Heat shock pretreatment could prevent the apoptosis of rat lymphocyte after H2O2 treated. Heat shock pretreatment could avoid rat lymphocyte from mitochondrial membrane potential depolarization, and subsequent cytochrome c release into cytoplasm after H2O2 treatment. The translocation of Bcl-2 into mitochondria of rat lymphocyte increased after H2O2 treatment in heat shock pretreated rats. We suggest that heat shock pretreatment contribute in protecting the septic subject from cell apoptosis by inhibiting the depolarization of mitochondrial membrane potential.

並列關鍵字

sepsis ； mitochondrial membrane potentail ； apoptosis ； Hsp72

參考文獻

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