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研究生:許庭瑀
研究生(外文):Tingyu Hsu
論文名稱:探討不同程度的缺氧對頭頸部鱗狀癌細胞增生之影響及自由基所扮演的角色
論文名稱(外文):The effects of different hypoxic conditions on proliferation of head and neck squamouse carcinoma cell lines and the role of reactive oxygen species
指導教授:康柏皇
指導教授(外文):BorHwang Kang
學位類別:碩士
校院名稱:國防醫學院
系所名稱:海底醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:67
中文關鍵詞:缺氧細胞增生活體外活性氧
外文關鍵詞:hypoxiacell proliferationin vitroROSBrdU
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頭頸部癌症是全世界最常見的第六大癌症,也是國內十大主要癌症之一,依據行政院衛生署九十七年公布的統計資料,口腔癌為男性十大癌症死因之第四位。頭頸部腫瘤之病生理尚不清楚,雖有早期診斷及多重治療方式,過去三十年來整體五年之存活率並未有實質改善。近年之研究顯示缺氧與腫瘤細胞的增生及侵犯性增加有關。先前之研究顯示缺氧會誘發活性氧分子的生成,而活性氧分子可以誘發訊號傳遞因子。在活體外癌細胞之培養系統中,缺氧可誘發腫瘤細胞之DNA合成增加,但未有確切證據指出細胞總數增加。本實驗室先前的研究亦顯示,1%氧氣暴露的確可誘發頭頸癌細胞DNA之合成,但卻抑制癌細胞之生長,使細胞總數較正常氧組為低。因此,本研究主要目的在於調整缺氧程度及培養條件建立缺氧誘發頭頸部鱗狀癌細胞增生之活體外模式,以供日後探討相關之分子機轉。自由基在此模式中所扮演的角色亦一併探討。我們利用兩株頭頸部鱗狀癌細胞株(FADU、SCC25)進行研究,實驗分成去血清(serum free)前處理24小時及沒有前處理兩組,並分別暴露於1%及3%氧氣或正常氧氣濃度下,並在不同的時間點收集細胞。以血球計數器計算細胞總數及BrdU嵌入試驗偵測細胞DNA合成之情形。利用nitro blue tetrazolium測量超氧自由基,並以2,7-dichloridihydrofluorescein diacetate測量過氧化氫之生成。去血清前處理下,1%氧氣暴露會抑制頭頸癌細胞之成長曲線, 3%氧氣暴露減輕此抑制現象。未經去血清前處理之情形下,1%及3%氧氣暴露可顯著誘發頭頸癌細胞之生長及DNA合成。缺氧亦會造成FADU和SCC25細胞內過氧化氫之增加,但是沒有發現超氧自由基的增加。FADU細胞以觸酶(Catalase)前處理後,細胞增生之現象受到抑制。本研究建立了缺氧誘發頭頸癌細胞增生之模式,且發現活性氧分子可能在此過程中扮演重要的角色。
Malignant disease is among the leading causes of mortality. Head and neck cancer is the sixth most common cancer in the worldwide. Oral cavity cancer ranked fourth among all cancer deaths of male in Taiwan. Despite of early diagnosis and multi-therapeutic modalities, the overall survival of patients head & neck cancer has not been improved substantially in the last three decades. Recent studies suggest that tumor hypoxia is associated with tumor cell proliferation and invasion. Hypoxia was known to induce production of reactive oxygen species (ROS), which may serve as signaling molecules and/or induce signal transduction. In vitro studies had shown that hypoxia induced DNA incorporation, an implication of increased cellular proliferation. However, there was no actual data showing increased cell number during hypoxia exposure. Our laboratory had previously found that 1% O2 exposure did induce DNA incorporation, however, it inhibited cell growth curve as compared to normoxia group. The current study was aimed to establish an in vitro cell model of hypxia-induced cancer cell proliferation to facilitate the study of underlying mechanisms. The possible involvement of ROS in this process was also investigated. Two head and neck squamous carcinoma cell lines, FADU and SCC25, were cultured in 1% O2, 3% O2, or room air for different periods of time. Cell number was determined by a hemacytometer after trypan blue staining. BrdU assay was applied for detecting DNA incorporation, an indicator of cell proliferation. The levels of superoxide anion and hydrogen peroxide were measured by nitro blue tetrazolium, and by 2,7-dichloridihydrofluorescein diacetate, respectively. The results showed 1% O2 exposure plus serum free pretreatment significantly inhibited cell growth curve. Three percent O2 had less inhibitiory effect. In cells without the serum free pre-treatment, 1% and 3% O2 enhanced cell growth, and BrdU uptake. Increased production of hydrogen peroxide, but not SOD, was noted in both FADU and SCC25 cell lines. When pretreated with catalase, the hypoxia induced FADU cell proliferation was inhibited. In summary, this study has established that 1% or 3% O2 exposure without serum-free pretreatment may stimulate proliferation of head and neck cancer cells in vitro. ROS, especially the hydrogen peroxide, may have an important role in hypoxia induced cancer cell proliferation.
目錄 ……………………………………………………………………… Ⅰ
圖次 ……………………………………………………………………… Ⅱ
中文摘要 ………………………………………………………………… Ⅴ
英文摘要 ………………………………………………………………… VII
第一章 緒論 ……………………………………………………………… 1
第二章 研究目的 ………………………………………………………… 9
第三章 實驗方法與材料 ………………………………………………… 10
第四章 實驗結果 ………………………………………………………… 19
第五章 結論 ……………………………………………………………… 34
第六章 討論 ……………………………………………………………… 35
第七章 參考文獻 ………………………………………………………… 40
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