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研究生:簡伊君
研究生(外文):Yi-Jun Jian
論文名稱:探討修補蛋白MSH2表現在熱休克蛋白90抑制劑增加pemetrexed誘發人類肺癌細胞毒性的角色
論文名稱(外文):Investigation the role of MSH2 expression in Hsp90 inhibitor enhancing pemetrexed-induced cytotoxicity in human lung cancer cells
指導教授:林芸薇
指導教授(外文):Yun-Wei Lin
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生化科技學系研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:103
語文別:中文
中文關鍵詞:非小細胞型肺癌愛寧達修補蛋白MSH2熱休克蛋白90p38MAPK
外文關鍵詞:NSCLCPemetrexedMuts homolog 2Heat shock protein 90p38MAPK
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多標靶的抗葉酸藥物pemetrexed (Alimta®﹔愛寧達)已被證實可用於臨床上治療非小細胞型肺癌。然而,有一些臨床試驗指出pemetrexed在治療上的效益又以非鱗狀細胞型態的非小細胞肺癌最為常見。到目前為止,仍然沒有合適的生物標記可用於臨床上來預測pemetrexed的治療效用。DNA的修復功能在鱗狀細胞癌對於化療的敏感性上扮演著關鍵的角色,其中人類MutS homolog 2(MSH2)是高度保留的DNA錯配修補系統中一個關鍵分子。到現在為止,pemetrexed是否會影響MSH2在非小細胞肺癌的表現仍是未知的。因此我們推測,向上調節MSH2的表現可能會促使細胞對pemetrexed產生抗藥性。另一方面,熱休克蛋白90的過度表現已被認為和非小細胞肺癌患者的治療預後不良有關,此外,抑制熱休克蛋白90的功能可同時影響多種致癌分子,並在體外和體內的試驗中呈現明顯的抗腫瘤作用。因此,在此研究中,主要探討是否藉由使用熱休克蛋白90的抑制劑來向下調控MSH2的表現可提高pemetrexed對人類非小細胞肺癌細胞的毒性。首先,觀察 pemetrexed使用在非小細胞肺癌細胞時對其MSH2 mRNA及蛋白質的影響。結果發現,當使用該藥物在20-50毫莫耳濃度時,MSH2 mRNA和蛋白質的表現會呈現一個MKK3 / 6-p38MAPK訊號活化依賴性的上升趨勢。此外,不論是轉染專一性的siRNA來抑制MSH2的表現,還是使用p38 MAPK抑制劑SB202190或siRNA來阻斷p38 MAPK的活化,都增強了pemetrexed對細胞的毒性。當將熱休克蛋白90抑制劑(17-AAG)與pemetrexed共同處理細胞時,更進一步地增強了pemetrexed誘導的細胞毒殺作用,並伴隨著MSH2蛋白與mRNA的降低。反之,在非小細胞肺癌細胞共同處理pemetrexed和熱休克蛋白90抑制劑之下,轉染持續活化態的MKK6E或HA-p38 MAPK基因載體後,恢復了p38 MAPK 的活性及MSH2蛋白和mRNA的表現與細胞的存活。此外,促使MSH2過度表現也抑制了pemetrexed和17-AAG共同處理之下對細胞的毒性。因此,根據本研究數據顯示,使用pemetrexed和熱休克蛋白90抑制劑共同治療,或抑制MSH2表現可作為非小細胞肺癌患者克服pemetrexed抗藥性的另一個新治療策略,雖然這些發現在臨床上的應用還有待更進一步人體試驗去證實。
Contents
Abbreviation 2
Background 4
Lung cancer 4
Pemetrexed 6
MSH2 9
p38 MAPK 11
Hsp90 13
Hsp90 inhibitor/ 17-AAG 14
Motive 15
Materials and Methods 16
Cell lines and reagents 16
Western blot analysis 16
Immunoprecipitation 17
Transfection with small interfering RNA 18
Transfection of expression plasmids into NSCLC cells 18
Cell viability analysis 18
Combination index analysis 19
Reverse transcriptase-polymerase chain reaction (RT-PCR) 19
Quantitative real-time PCR 20
Determination of cell death and growth inhibition 20
Colony-forming ability assay 20
Statistical analysis 21
Aims 22
Results 23
Discussion 29
References 33
Figures and Legends 41
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