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研究生:陳臻
論文名稱:自由基對口腔癌細胞的毒殺作用
論文名稱(外文):Cytotoxicity of Free Radicals on Oral Carcinoma Cell Line
指導教授:陳振漢陳振漢引用關係
學位類別:碩士
校院名稱:國防醫學院
系所名稱:牙醫學系
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:69
中文關鍵詞:自由基口腔癌
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  本研究希望建立一個活體外(in vitro)的實驗模式,探討自由基對口腔癌細胞的影響,以瞭解其生物特性。我們以SNAP(NO供給者)及H2O2處理口腔癌細胞株OCE-MI後,結果能引發細胞死亡,而死亡程度隨濃度及作用時間增加而增加。再經由加入NO及H2O2清除劑拮抗後,確認細胞死亡是經由NO及H2O2所引起。由於癌細胞經常處於發炎環境中,因此我們想瞭解,免疫細胞所釋出的細胞激素對於自由基的細胞毒性有何影響,當OEC-MI細胞分別以細胞激素(TNF-□、TGF-□1、IL-1□、IFN-□)預先處理過後,再與NO及H2O2作用,結果OEC-MI細胞對NO及H2O2的細胞毒性更為敏感,由此結果看來,細胞激素應可補助自由基的毒殺作用。
  分析NO及H2O2所引發OEC-MI 細胞的死亡方式後,我們發現NO及H2O2均能引發OEC-M1細胞產生凋亡及壞死二種死亡方式。利用acridine orange染色法觀察細胞形態的變化,發現NO與H2O2所造成的細胞死亡形態不太相同,NO可以誘發OEC-MI細胞產生明顯凋亡現象,而H2O2則不易觀察到明顯的凋亡細胞特徵。
  探究細胞凋亡相關的基因調控因子,發現當H2O2和SNAP的作用時間及濃度增加時,抗凋亡蛋白Bc1-2的表現無明顯變化,而凋亡促進因子p53的表現不增反減,而能與Bc1-2結合凋亡促進蛋白Bax則是明顯逐漸增強。因此推測當OEC-MI細胞受NO及H2O2毒殺時,Bax在引發細胞死亡過程中扮演著一個角色。
  由於OEC-M1細胞本身明顯表現Bc1-2蛋白,我們分析了CHO和CHO-Bc1-2細胞在H2O2和SNAP作用下的死亡方式後,發現當Bc1-2無法抵擋H2O2的毒殺時,CHO-Bc1-2細胞會以凋亡方式死亡,而無Bc1-2的CHO細胞則主要以壞死方式死亡;Bc1-2似乎對NO引發的細胞死亡方式沒有太大的影響。
  綜合我們的研究結果,我們認為未來可考慮運用自由基來治療口腔癌,藉由引發口腔癌細胞產生細胞凋亡。


  We wish to establish an in vitro model of the effects of free radicals on oral cancer cells. We used oral cancer cell line, OEC-M1, as target cells, and SNAP and H2O2 as free radical donors. SNAP and H2O2 induced time- and dose-dependent cell death of OEC-M1. The NO scavenger, oxyhemoglobin, can inhibit the cytotoxicity of NO, and the H2O2 scavenger, catalase, can inhibit the cytotoxicity of H2O2, too.
  In vivo, tumor cells often infiltrate in the inflammation. We want to know the corelation between the cytokines and free radicals. After preincubation with the cytokines (TNF-□, IL-1□, TGF-□1 , IFN-□) for 48 hours, the cells were treated by H2O2 and SNAP. The results were that the OEC-M1 cells were more sensitive to the cytotoxicity of NO and H2O2, after cytokines pretreatment. So the cytokines could enhance the cell death.
  By cell death detection ELISA kit and LDH kit, we discovered that NO and H2O2 both can induced apoptosis and necrosis of OEC-M1 cells. Acridine orange staining microscopic examination revealed that cellular morphology of OEC-M1 was different between NO and H2O2 treatment. It was easy to detect NO-induced apoptotic morphology, but it was not easy to detect H2O2-induced apoptotic morphology.
  Apoptosis was regulated by several gene proteins. We discovered that expression of anti-apoptosis protein, Bcl-2 didn't change, and the apoptotic proteins, p53 and Bax was decreased when cell death induced by NO and H2O2. We suggest that Bax maybe a key factor of cell death induced by NO and H2O2.
  To understand the Bc1-2 function, we analyzed the death type of CHO and CHO-Bcl-2 cells induced by H2O2 and NO. We discovered that CHO-Bcl-2 went on a process of apoptosis when treated with high dose H2O2, but the CHO cells were necrosis. As to the cytotoxicity of NO, Bcl-2 seemed to have no protective effect of cells.
  According to these data, we consider that free radicals treatment maybe a useful method to do with oral cancer cells by induction of apoptosis.

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