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研究生:鄭自君
研究生(外文):Zi-Jun Zheng
論文名稱:在酒精長期刺激人類肝癌細胞株Hep3B的模式中,探討酒精誘發Tissuetransglutaminase表達的分子機制
論文名稱(外文):Study on the ethanol long-term stimulating Hep 3B model for examining the molecular mechanism of ethanol- induced tissue transglutaminase expression.
指導教授:何元順
指導教授(外文):Yuan-Soon Ho
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗生物技術學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:87
中文關鍵詞:酒精肝癌氧化壓力
外文關鍵詞:alcoholHCCoxidative stressTGM2
相關次數:
  • 被引用被引用:1
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  • 下載下載:68
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本研究主要探討在酒精長期處理的Hep 3B細胞株中,酒精誘發tissue transglutaminase(TGM2)表達的分子機制。TGM2具有多種功能,包括Ca2+-dependent transamidation,以催化蛋白質之間的cross- linkage,穩定細胞外基質蛋白或造成纖維化的發生;另外一個功能為 GTPase protein,作為訊息傳遞分子。在本研究中,比較不處理酒精的Hep 3B細胞株和酒精(0.06%)長期處理的Hep 3B細胞株時,可以發現在酒精長期處理的Hep 3B細胞株,其TGM2的mRNA和蛋白質皆有穩定的表現。在酒精長期處理下,欲知影響TGM2轉錄的轉錄因子,而文獻指出,NF-kappaB參與調控TGM2的表現,此外亦有文獻指出,受到酒精刺激,AP1、SP1會被活化並轉位到細胞核內,進而調控目標基因的表現。因此利用chromatin immunoprecipitation和核質分離,證明在酒精長期處理的Hep 3B細胞株中,調控TGM2 mRNA表現的轉錄因子為AP1、SP1,並非NF-kappaB。文獻指出,酒精會透過MAPK作為訊息傳遞路徑,特別是ERK pathway,在酒精長期處理的Hep 3B細胞株中,本研究證實酒精不是透過ERK的訊息傳遞路徑來誘導TGM2表現。
The purpose of this investigation is to study the molecular mechanism of ethanol-induced tissue transglutaminase expression in ethanol long term- treated Hep 3B cells. TGM2 is a multifunctional enzyme. TGM2 is a Ca2+- dependent enzyme which plays an important role in the stabilization of the extracellular matrix (ECM) proteins and formation of fibrosis by catalyzing protein-protein cross-linkage. TGM2 also is a GTPase protein. In this investigation, mRNA and protein levels of TGM2 can express stablely in ethanol long term-treated Hep 3B cells. Recent works have shown that transcription factor NF-kappaB was required to regulating TGM2 expression. Another recent works have shown that transcription factors AP1 and SP1 are activated and translocated into nucleus for regulating gene expression when exposing ethanol. Using chromatin immuno- precipitation and isolation of nucleus and cytosolic extracts, we found that transcription factors regulating TGM2 expression are AP1 and SP1, not NF-kappaB in ethanol long term-treated Hep 3B cells. Recent works have shown the ethanol-induced signal transduction go through MAPK pathway, especially ERK pathway. We found the signal transduction pathway of regulating TGM2 expression isn''t go through ERK pathway in ethanol long term-treated Hep 3B cells.
目錄(contents)
論文摘要1
Abstract3
目 錄(Contents)5
圖 目 錄(Figure and Talbes lists)8
縮 寫 表(Abbreviations)9
第一章 緒論(Introduction)13
前言 14
(一)肝臟疾病簡介14
(二)酒精與其相關疾病16
(三)肝纖維化相關基因18
(四)Transglutaminase(TG)family介紹18
(五)Tissue transglutaminase(TGM2)介紹20
(六)NF-kappaB pathway的簡介21
(七)MAPK pathway的簡介 22
(八)PI3K/Akt pathway的簡介25
研究目的 25
第二章 實驗材料與方法(Materials and Methods)28
(一) 藥品與試劑 29
(二) 常用溶劑32
(三) 實驗方法34
1. 細胞株和細胞培養34
2. 培養酒精長期處理的人類肝癌細胞株Hep 3B cells35
3. 反轉錄-聚合酶連鎖反應(RT-PCR)35
4. 西方式點墨法和核質分離37
5. Chromatin immunoprecipitation(ChIP)39
6. 氧化壓力的檢測41
7. Protein oxidation assay41
8. MTT assay42
第三章 實驗結果(Results)44
一、酒精對於人類肝癌細胞株Hep 3B cells中誘發TGM2表現的情形45
二、酒精對於人類肝癌細胞株Hep 3B cells外觀的影響46
三、酒精誘發TGM2表現,以探討與TGM2 promoter鍵結的transcription factors 47
四、在酒精長期處理的Hep 3B cells中,調控TGM2表現的轉錄因子為SP1和AP1,而非NF-kappaB47
五、隨著酒精處理的時間越長,轉錄因子轉位到細胞核內的情況越明顯48
六、酒精造成細胞的reactive oxygen species(ROS)增加,使得氧化壓力增加,造成細胞的傷害49
七、在酒精長期處理的Hep 3B cells中,調控TGM2的表現並非透過ERK pathway 50
第四章 討論(Disccusion)52
第五章 圖表(Figures)60
第六章 文獻參考(References73
附錄(Appendices)81
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