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研究生:林宜瑩
研究生(外文):Yi-Ying Lin
論文名稱:雷公藤內酯醇調控轉錄因子Nrf2與HO-1表現之探討
論文名稱(外文):Triptolide Inhibits Heme Oxygenase-1 Expression Via Nrf2 Signaling Pathway
指導教授:徐慧雯徐慧雯引用關係
指導教授(外文):Huey-Wen Shyu
學位類別:碩士
校院名稱:輔英科技大學
系所名稱:醫事技術系碩士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:66
中文關鍵詞:p38p53Nrf2雷公藤內酯醇第一型血色素氧化酵素
外文關鍵詞:p53Nrf2p38Heme oxygenase-1Triptolid
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第一型血色素氧化酵素 (Heme oxygenase 1, HO-1)廣泛存在於腫瘤細胞內,是一種具有細胞保護作用的蛋白質,其功能涵蓋了抗氧化及抗凋亡作用,並可減少活性氧化物質對細胞的傷害,因此可以促進腫瘤細胞增生。雷公藤內酯醇 (triptolide)是由雷公藤根部萃取純化所得的成分之一,已應用於抗腫瘤與免疫抑制的研究。本論文中主要是針對triptolide在肺腫瘤細胞的傷害途徑進行探討。
研究結果顯示triptolide可抑制多種腫瘤細胞的生長,例如肺癌。其抑制作用呈現時間和劑量之效應。同時,在肺癌A549細胞中訊息傳導路徑的p38蛋白質其活化明顯受到triptolide之抑制。
藉由RT-PCR及西方墨點法分析結果證明triptolide會造成腫瘤細胞中之HO-1 mRNA與蛋白表現大量減少,並可誘發細胞核中Nrf2轉位進細胞質。進一步利用冷光酶試驗系統觀察HO-1啟動子的表現,確認triptolide對HO-1之抑制作用是透過Nrf2的結合位置調控。此外,使用染色體免疫沉澱實驗分別證明在Nrf2或p53蛋白皆可結合在HO-1啟動子上,triptolide可以藉由減少Nrf2在HO-1啟動子上的結合與增加p53在HO-1啟動子上之結合來調控HO-1活性。
本研究結果顯示,腫瘤細胞的增生是藉由Nrf2與p53來調控HO-1的表現。綜合上述結果,我們認為triptolide對HO-1表現量之影響與肺癌細胞的生長具關聯性,此結果可為防治肺癌提供一條新的思路。
Heme oxygenase-1 (HO-1) is expressed in various tumor cells and plays an important role in tumor cell growth through anti-oxidative and anti-apoptotic effects. It has been reported that inhibition of HO-1 expression induced apoptosis. Triptolid, a major active component extracted from the root of Triptergium wilfordii hook. f. (TWHF), has been shown to be a potent anticancer agent. However, the effect of triptolid on lung cancer cells remains unknown. In this study, we observed that triptolid treatment decreased the cell viability of A549 cells in a dose dependent manner.
To identify whether the mitogen-activated protein kinase (MAPK) signalling pathway is involved in the triptolid induced cytotoxicity in A549 cells, the phosphorylated forms of ERK1/2, p38, JNK were determined. Results showed that the phosphorylated p38 was attenuated in the triptolid-treated A549 cells. In addition, both the RT-PCR and western blot analysis demonstrated that the expression levels of HO-1 were inhibited in the triptolid-treated A549 cells. The suppression of HO-1 expression in triptolid-treated A549 cells, was modulated by the nuclear translocation of Nrf2. Previous studies have suggested that HO-1 is a direct p53 target gene. Further to investigate the effect of p53 on the regulation of HO-1 expression in triptolid-treated A549 cells, chromatin immunoprecipitation experiments were performed. Results indicate that p53-dependent trans-repression is due to the direct interaction of p53 with the ARE-containing promoters.
These data suggest that the inhibition of HO-1 expression may involve in the triptolid induced cytotoxicity in lung cancer A549 cells, and the decrease of HO-1 is mediated by its upstream regulators, Nrf2 and p53.
誌 謝 (Acknowledgements) i
中 文 摘 要 (Abstract in Chinese) ii
英 文 摘 要 (Abstract in English) iii
目 錄 (Contents) iv
圖 目 錄 (Figure Contents) vi
縮 寫 檢 索 表 (Abbreviation) viii
第一章、緒論 (Introduction) 1
第一節、雷公藤之簡介 1
第二節、第一型血紅素氧化酶之結構、表現與功能 3
第三節、Nrf2於調控基因轉錄作用之角色 7
第四節、抑癌基因p53的結構、表現與生理功能 8
第五節、細胞內信號傳導機制 9
研 究 目 的 與 動 機 11
實 驗 設 計 12
第二章、實驗材料與方法 (Materials and Methods) 13
第一節、儀器 13
第二節、試劑藥品暨耗材 15
第三節、實驗方法 17
一、細胞培養 (Cell Culture) 17
二、細胞存活分析 (Cell Viability Assays) 17
三、萃取細胞RNA (RNA Isolation) 18
四、反轉錄反應 (Reverse Transcription, RT) 18
五、聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 19
六、免疫螢光染色 (Immunofluorescence Assay, IFA) 19
七、西方墨點分析法 (Western Blotting Analysis) 20
八、載體構築 (Plasmid Construction) 21
九、質體轉形 (Transformation) 22
十、質體DNA的製備 (Plasmid DNA Extraction) 22
十一、暫時性表現共同轉染分析 (Transient Cotransfection Assay) 23
十二、染色質免疫沉澱實驗 (Chromatin Immunoprecipitation, ChIP) 25
第三章、實驗結果 (Results) 27
第一節、Triptolide處理後細胞存活現象 27
第二節、Triptolide處理後HO-1變化的情形 27
第三節、Triptolide藉由HO-1啟動子調控HO-1表現 28
第四節、Triptolide抑制Nrf2的基因表現 29
第五節、Triptolide對mitogen activated protein kinase (MAPK)與AKT的影響 30
第六節、Triptolide誘引A549細胞與HepG2 中p53蛋白質表現 31
第四章、結 論 與 未 來 展 望 (Conclusion and Perspective) 33
第一節、結論 33
第二節、未來展望 35
圖 (Figures) 36
參 考 文 獻 (References) 59
著 作 發 表 與 獎 項 (Publications and Honors) 64
附 錄 66
附錄一:進行PCR使用之引子序列 66
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