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研究生:洪千雯
研究生(外文):Chien-Wen Hung
論文名稱:Hepa-1小鼠細胞蛋白質體如何受2,3,7,8-四氯戴奧辛影響
論文名稱(外文):The Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) Treatment on Mouse Hepa-1 Cell Proteome
指導教授:廖寶琦廖寶琦引用關係
指導教授(外文):Pao-Chi Liao
學位類別:碩士
校院名稱:國立成功大學
系所名稱:環境醫學研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:99
中文關鍵詞:液相層析串聯式質譜儀蛋白質體Hepa-1 細胞TCDD二維膠電泳
外文關鍵詞:proteomicHepa-1 cellliquid chromatography-tandem massTCDD
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戴奧辛(dioixins)是一系列廣泛存在的環境污染物,具有高度毒性與生物蓄積性,對許多生物的健康造成危害。在眾多戴奧辛的同源物(congeners)中,以2,3,7,8-四氯戴奧辛(2,3,7,8-tetrachlorodibenzo-p-dioxin,簡稱TCDD)的毒性最強,毒性反應包含致癌性、皮膚毒性、肝臟毒性、免疫毒性與生殖毒性等。對於戴奧辛的毒性機制,以往的研究認為與芳香基碳氫化合物受體(arylhydrocarbon receptor-AhR)的活化有關;TCDD 活化了AhR,被活化的AhR 從細胞質轉移到細胞核內,在細胞核與另一個蛋白質AhR nuclear translocator(ARNT)形成二聚體(heterodimer),並與DNA 上的xenobiotic regulatory element(XRE)結合,誘發某些基因(如:CYP1A1)的轉譯與表現;不過,單從AhR的代謝途徑並不能解釋所有生物上的毒性現象。而二維膠電泳(two-dimensionalgel electrophoresis,簡稱2-DE)的優點則是可以在一次實驗裡,同時對許多蛋白質的變化做觀察。本研究以2-DE 的方法,觀察小鼠肝癌細胞株Hepa-1 的蛋白質體,是否會受TCDD 的影響而改變。並且,評估一個合適的統計方法分析這些蛋白質量化的數據。2-DE 以銀染方式顯色,解析出約800 個蛋白質,在六次2-DE 的重複試驗結果,不同次實驗之間2-DE 的變異性很大,但同一次實驗的實驗組(10 nM TCDD,20 hr)與對照組(DMSO)的2-DE 幾乎完全相同。所以要比較實驗組與對照組的2-DE,應該將同一次的兩片膠視為一個配對(pair),使用paired-t test 分析蛋白質量的變化是否達統計上顯著差異。經paired-t test 統計分析,大部分的蛋白質量的變化在實驗組與對照組中都沒有顯著差異,但十個蛋白質(九個增加、一個減少)量的變化達統計上顯著差異(P<0.05)。選擇十個有統計上顯著差異的其中的七個蛋白質以液相層析串聯式質譜儀鑑定,發現這七個蛋白質的其中三個與蛋白質合成或分解相關( ribosomal protein 與proteasome),一個與信息傳遞有關(RhoGDI-1),兩個細胞的氧化還原反應(peroxinredoxin 與thioredoxin-related protein mRNA),還有一個是常見的酵素(glutamate-cysteine ligase regulatory subunit gene)。這些蛋白質與TCDD 毒性機制的關係值得在未來加以研究證明。
Dioxins are structurally related environmental pollutants. They bioaccumulate via food chain and cause a variety of health effects, including carcinogenesis, skin-, hepato-, immuno- and reproductive toxicity. Among 75 dioxin congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic one. In response to TCDD exposure, AhR nuclear translocator (ARNT) forms a heterodimer with the aryl hydrocarbon receptor (AhR) after the ligand-dependent translocation of AhR to the nucleus, and eventually the complex binds to the xenobiotic response elements (XREs), which promote transcription of several specific genes, such as CYP1A1.However, the diverse effects of TCDD could not be simply explained by the AhR pathway. The objective of this study is to use proteomic approaches to investigate the overall protein profile in mouse Hepa-1 cells stimulated by TCDD. Quantified protein spots revealed by two-dimensional gel electrophoresis (2-DE) were evaluated statistically to study the effect of dioxin treatment. Usually, more than 800 spots were detected on each 2-DE gel. The between-run variations of silver-stained protein signals were found to be much greater than within-run variations when 2-DE were performed using a multiple-gel system. Therefore, paired-t test was used to compare 2-DE data derived from TCDD treated-cells (10nM TCDD, 20 hr) and carrier controls. Among 865 protein spots analyzed, the abundances of nine proteins were found to increase while only one protein showing lower abundance (P<0.05). 7 of the 10 significant regulated protein spots were chosen for subsequent identification by high performance liquid chromatography-tandem mass spectrometry. The 7 proteins were identified as glutamate-cysteine ligase regulatory subunit gene, Gdi-1 mRNA for RhoGDI-1, proteasome subunit beta type 6 precursor, proteasome subunit beta type 4 precusor, 40s ribosomal protein, thioredoxin xidase, thioredoxin-related protein mRNA and elongation factor 1-delta. Three proteins were classified as “biosynthesis” and “biodegradation” proteins (ribosomal protein and proteasome). The “RhoGDI-1” might be involved in signal transduction pathway. Two proteins (peroxinredoxin and thioredoxin-related protein mRNA) might play important roles in eliminating peroxides generated during metabolism.
主目錄
中文摘要………………………………………………………………I
英文摘要………………………………………………………………II
表目錄…………………………………………………………………IV
圖目錄…………………………………………………………………V
中英文對照表…………………………………………………………VI
英文縮寫對照表………………………………………………………VII

第一章 序論……………………………………………………………1
  第一節 研究動機…………………………………………………2
  第二節 研究目的…………………………………………………3
第二章 文獻探討………………………………………………………4
  第一節 戴奧辛簡介………………………………………………5
  第二節 蛋白質體學………………………………………………10
第三章 研究方法與材料………………………………………………14
  第一節 研究架構…………………………………………………15
  第二節 使用試劑與常用溶液配方………………………………16
  第三節 研究方法…………………………………………………19
第四章 結果與討論……………………………………………………32
  第一節 研究結果…………………………………………………33
第二節 討論……………………………………………………………47

結論 ……………………………………………………………………51
參考文獻 ………………………………………………………………53
附錄一 …………………………………………………………………58
附錄二 …………………………………………………………………61
附錄三 …………………………………………………………………83
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