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研究生:呂怡慧
研究生(外文):Lu Yi-Huey
論文名稱:鎵鍺銦銻對於NIH/3T3細胞致毒機制之研究
論文名稱(外文):Studies on the cytotoxic mechanisms of Gallium, Germanium, Indium and Antimony in NIH/3T3 cells
指導教授:呂鋒洲呂鋒洲引用關係
指導教授(外文):Lu Fung-Lu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化學研究所
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:1999
畢業學年度:87
語文別:中文
論文頁數:70
中文關鍵詞:細胞凋亡自由基重金屬
外文關鍵詞:apoptosisfree radicalheavy metalNF-kappa B
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摘 要
電子工業以鎵、鍺、銦、銻為新的半導體材料,來達成更快速、安定、輕巧的經濟及商業上的訴求。由於這些金屬成份特殊的化性,其廢棄物及副產物往往造成環境處理上的負擔,由臨床統計數字顯示,此類重金屬的無機代謝物將對人體造成嚴重損傷。
本研究主要以硫酸鎵 (Ga2(SO4)3) 、二氧化鍺(GeO2)、氯化銦 (InCl3)、氯化銻 (SbCl3) 四種金屬化合物處理老鼠纖維母細胞株(NIH/3T3 cell line),觀察這四種重金屬對於細胞毒性的作用情形,及其是否促使活性氧物種 (如過氧化氫) 產生,是否誘發轉錄因子NF-B產生。
將四種重金屬處理老鼠纖維母細胞株NIH/3T3後,經由MTT assay測細胞毒性,在72小時後,四種重金屬對細胞均有一定程度的毒性,並且與濃度呈正相關。再經由流式細胞儀(Flow cytometry)測定細胞產生過氧化氫(H2O2)的情形,將四種重金屬處理細胞2小時後,發現氯化銦、氯化銻會促使細胞產生過氧化氫;4小時後,硫酸鎵也有相同的現象。將100g/ml的氯化銻處理細胞後,抽細胞質及細胞核蛋白,利用西方點墨法(Western blot),偵測IB在不同時間表現量的變化情形,發現隨時間IB表現量有逐漸降低的現象;再利用NF-B的oligonucleotidic probe,偵測NF-B表現量的變化情形,發現NF-B表現量有逐漸增加的現象。NF-B的表現與H2O2是否有直接的關係,則需進一步加入抗氧化物質來確認。
此外,在10、50及100g/ml濃度的氯化銻處理後,抽DNA,利用瓊脂凝膠電泳,觀察是否有DNA斷裂的情形,發現氯化銻可促使細胞產生階梯式斷裂,以PI染色,利用流式細胞儀偵測DNA分佈的情形,發現在氯化銻的作用下,DNA產生嚴重斷裂的現象,利用光學顯微鏡及螢光顯微鏡觀察經氯化銻處理後的細胞,其外型出現萎縮及染色質濃縮現象。以上觀察到的現象可被抗氧化物質catalase所抑制,因此,我們發現,活性氧物種在氯化銻誘發細胞發生凋亡時扮演一重要角色。

The research on metals for use in semiconductors had been accompanied by an increasing amount of toxic materials released as potential toxic wastes, which were harmful to health and environment. The purpose of this study was to clarify the molecular mechanism of the cytotoxicity of these semiconductor-metals including gallium, germanium, indium and antimony.
After treatment of NIH/3T3 cells with Ga2(SO4)3, GeO2, InCl3, and SbCl3 for 72 hours, we found that these metal ions were toxic to cells, and showed dose-dependent response of cytotoxicities which were measured by MTT assay. Exposure of NIH/3T3 cells to InCl3 (100 g/ml) and SbCl3 (100 g/ml) for 2 and 4 hours leaded to the generation of hydrogen peroxide, measured with the DCFH-DA regent to the cells. Exposure to SbCl3 (100 g/ml) with NIH/3T3 cells for 2 and 4 hours could induce the activation of NF-B using the electrophoretic mobility shift assay (EMSA).
Treatment of NIH/3T3 cells with SbCl3 resulted in the induction of apoptosis as shown by the DNA fragmentation and morphological changes. The DNA fragmentation due to apoptosis could be blocked by catalase (an inhibitor of H2O2). suggesting that induction of apoptosis of NIH/3T3 cells by SbCl3 may be mediated through reactive oxygen intermediates generated by SbCl3. In conclusion, this data shows that ROI might act as one of messengers in induction of apoptosis of NIH/3T3 cells.

中文摘要…………………………………………………………..1
英文摘要…………………………………………………………..3
前言………………………………………………………………..5
縮寫表…………………………………………………………….16
實驗儀器及材料………………………………………………….18
實驗方法及步驟………………………………………………...24
結果……………………………………………………………….38
討論……………………………………………………………….44
參考文獻……………………………………………………….…49
圖表…………………………………………………………….…59

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