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研究生:劉穎昇
研究生(外文):Liu,Ying-Sheng
論文名稱:HER-2/neu造成膀胱癌細胞株對methotrexate敏感性之分子機轉
論文名稱(外文):The molecular mechanisms of HER-2/neu induced sensitivity to methotrexate in bladder cancer cell lines
指導教授:賴明德賴明德引用關係
指導教授(外文):Lai, Ming-Derg
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:英文
論文頁數:89
中文關鍵詞:酪氨酸激脢膀胱癌抗藥性訊息傳遞
外文關鍵詞:Her-2/neutyrosine kinasebladder cancermethotrexatedrug resistantapoptosisERK mapkp38 mapk
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摘 要
Her-2/neu 致癌基因屬erbB族群的成員之一,其蛋白質產物為185kDa的穿膜受體(transmembrane receptor) p185erbB2,具有酪氨酸激脢(tyrosine kinase)活性,對於控制細胞生長、分化等扮演重要角色,在特定癌細胞中,如非小球性肺癌(Non-small cell lung cancer)、乳癌即常發現neu的大量表現。臨床及基礎研究中指出此基因之大量表現常導致細胞對化學治療劑的藥物拮抗性。
本實驗室先前以含活化型(Val664→Glu)及原生型neu之質體轉染一株P185 erbB2表現量極低的膀胱癌細胞株TCCSUP,分別獲得兩株具活化態neu轉染細胞NA1、NA2及三株正常態之neu轉染細胞N5、N10、N14。在分析其對化學治療劑methotrexate之毒殺曲線時,發現正常態neu大量表現之轉染細胞具有明顯較高的藥物敏感性,並可能經由細胞凋亡機轉造成細胞死亡。
為探討細胞分子層次上,methotrexate造成HER-2/neu轉染細胞株死亡的可能機制,目前我們的研究著重於分析methotrexate處理下的轉染細胞株其內在HER-2/neu 下游各種訊息傳遞途徑(signal transduction)各主要的變化,我初步選定分析的對象包括: Mitogen-activated protein kinase superfamily中的 ERK MAPK激脢途徑(主要之訊息傳遞角色為細胞之生長及分化);p38激脢途徑(主要之角色為應付細胞對於外界壓力,藥物反應之相關基因表現)。實驗結果顯示(1)Neu之轉染細胞株的Ras/Raf/ERK MAPK途徑擁有較高基礎活性。(2)ERK MAPK活性在MTX處理時,可能和細胞生長密度(cell density)有關,正常態neu的轉染細胞在低細胞密度時ERK磷酸化不受MTX影響,高細胞密度時則呈少量磷酸化的反應。(3)ERK,p38 MAPK兩激脢途徑在參與MTX所造成neu轉染細胞株的細胞死亡途徑可能僅佔有非決定性影響力(Non-determining factor)。更進一步地,我們將繼續研究apoptosis之引發與其他訊息傳遞途徑之關係。

Abstract
HER-2/Neu, a proto-oncogene encodes a 185-kDa protein, which belongs to type I receptor tyrosine kinase family, and is essential for cell growth, differentiation regulation. Overexpression of Neu is frequently observed in many tumor types such as Non-small-cell-lung-cancer (NSCLC), breast cancer where it correlates with intrinsic chemotherapeutic resistance.
To investigate the role of Neu in bladder cancer, both wild-type and an active version of HER-2/Neu (Val664-to-Glu) transfectant were established in TCCSUP human bladder transitional carcinoma cell line previously, the cell cytotoxic characteristics to anti-cancer drugs of these transfectants were examined. We found that the chemosensitivity to methotrexate (MTX), but not cisplatin or adriamycin observed in TCCSUP-Neu transfectants is higher than both parental and control cell lines. The cell death of TCCSUP-Neu transfectants during MTX treat is prone to undergo apoptosis.
To understand the underlying mechanisms, we examined the major signal transduction pathways downstream HER-2/neu during MTX treatment, our results indicate that:
(1) TCCSUP-Neu transfectants contain high basal ERK MAPK activity. (2) The response of ERK MAPK to MTX may be dependent on cell density. ERK activation is only observed at confluent cell density. (3) The activation of ERK and p38 MAPK is not essential for MTX-induced cell death.

目 錄
一、 授權書
二、 考試合格證明
三、 誌謝感言
四、 目錄 1
五、 摘要
(1) 中文摘要 2
(2) 英文摘要 4
六、 縮寫表 5
七、 圖表錄 7
八、 緒論 9
九、 材料與方法 16
十、 結果 45
十一、 討論 54
十二、 參考文獻 59
十三、 圖 65
十四、 附錄 86
十五、 自述 88

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