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研究生:徐千婷
研究生(外文):Chien-Ting Hsu
論文名稱:單一活細胞表皮生長因子受體酪胺酸酶抑制劑PD153035作用之即時反應觀測
論文名稱(外文):Visualizing the Real-Time Response to EGFR Tyrosine Kinase Inhibitor PD153035 in Single Living Cells
指導教授:楊自森
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:生醫材料暨工程研究所
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:2010
畢業學年度:98
語文別:中文
論文頁數:74
中文關鍵詞:表皮生長因子受體酪胺酸&表皮生長因子受體酪胺酸&表皮生長因子受體酪胺酸&表皮生長因子受體酪胺酸&表皮生長因子受體酪胺酸&表皮生長因子受體酪胺酸&表皮生長因子受體酪胺酸&
外文關鍵詞:epidermal growth factorepidermal growth factor receptorepidermal growth factor receptor- tyrosine kinase inhibitorPD153035Quantum dots
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根據國民健康局統計數據顯示,2009年肺癌已躍升國人癌症死因之前三名。臨床上肺癌分為小細胞肺癌和非小細胞肺癌兩類,其中非小細胞肺癌發生率佔85-88%。一般非小細胞治療方式主要以化學治療和標靶藥物治療為主。由於大約88-99% 的非小細胞肺癌會出現表皮生長因子受體(epidermal growth factor receptor,EGFR)的大量表現,因此表皮生長因子受體酪胺酸酶抑制劑(epidermal growth factor receptor- tyrosine kinase inhibitor,EGFR-TKI)已為非小細胞肺癌理想標靶治療藥物。目前非小細胞肺癌第二、三線標靶治療之藥物主要有艾瑞莎(Iressa)及得舒緩(Tarceva),兩者皆為表皮生長因子受體酪胺酸酶抑制劑,相關研究證實EGFR-TKI會抑制EGFR之磷酸化反應,藉由阻斷受體將訊號傳入細胞核中,可以達到抑制腫瘤細胞增生的目的。
在過去文獻證實PD153035能夠阻斷表皮生長因子(EGF)誘導EGFR自體磷酸化(autophosphorylation)達到抑制大量表現EGFR之腫瘤細胞增生。本論文提出以生物單分子螢光技術探討PD153035如何影響肺癌細胞EGFR運動傳輸模式,以及進行PD153035抑制劑可否再由細胞膜內被移除的可逆性驗證。本論文同時也建立單一細胞層級生物單分子的偵測方法學,包括單分子影像追蹤系統、MSD統計分析方法以及速度能量頻譜分析等相關分析工具。本研究主要結論是(1)透過增加EGF濃度可以提高QD-EGF經由胞吞作用進入細胞內的能力:(2)在1 μM PD153035的濃度下可完整抑制A549細胞株EGFR的活性: (3)利用單分子技術證實PD153035與EGFR酪胺酸酶的ATP結合位點的結合過程是一個可逆反應: (4)完成QD-EGF於單一活細胞MG63的軌跡追蹤以及QD-EGF於細胞內部的擴散係數定量量測。


According to the statistics from Bureau of Health Promotion, lung cancer was the leading cause of cancer-related death in Taiwan in 2008. The main types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The occurrence of NSCLC accounts for as many as 85% to 88% of cases. The primary treatment for NSCLC usually involves chemotherapy and targeted therapy. Because the epidermal growth factor receptor (EGFR) is highly expressed in 88 to 99% of NSCLC, EGFR-tyrosine kinase has become a particularly promising targeted drug for treating NSCLC. Presently, Iressa and Tarceva are the second and third line treatment in NSCLC. All of them can inhibit the growth of cancer cells expressing high level of EGFR via blocking the EGF-induced activation of the EGFR tyrosine kinase.
According to previous studies, PD153035 can block the EGF-induced autophosphorylation of EGFR and inhibit the growth of cancer cells which expressing high level of EGFR. We used single-molecule fluorescence imaging technique to investigate the effect of PD153035 on the EGFR trafficking and investigate whether the EGFR could be reactivated when PD153035 leaches out from the lung cancer cells. In addition, we also established single-molecule diagnostic techniques at single cell level; this allows us to conduct single particle tracking, to determine the mean square displacement, and to calculate the power spectrum of velocity fluctuations. The primary conclusions of this thesis are summarized as follows. (1) The internalization of QD-EGF could be enhanced due to the presence of high concentration of free biotin-EGF; (2) The EGFR activity of A549 cell line could be completely inhibited due to the presence of 1 ?嵱 PD153035; (3) The inhibition of the EGFR tyrosine kinase activity due to PD153035 is reversable; (4) The trafficking of QD-EGF and the calculation of the diffusion coefficient of QD-EGF has been successfully determined for living MG63 cells.


中文摘要 Ⅰ
Abstract Ⅲ
致謝 Ⅴ
目錄 Ⅶ
表目錄 Ⅹ
圖目錄 ⅩⅠ
第一章 緒論 1
1-1前言 1
1-2研究動機與目的 2
1-3文獻回顧 4
1-3-1 肺癌介紹 4
1-3-1-A 肺癌診斷與分類 5
1-3-1-B 肺癌治療 8
1-3-2表皮生長因子介紹 10
1-3-3 表皮生長因子受體之訊息傳遞路徑 111
1-3-4 標靶藥物PD153035介紹 13
1-3-5 單分子螢光觀測技術介紹 15
1-4章節提要 19
第二章 實驗原理 21
2-1 單一細胞層級之單分子螢光影像技術 21
2-2 單分子影像追蹤技術 24
2-3 平均平方位移原理介紹 28
第三章 系統介紹 30
3-1單分子螢光觀測系統介紹 30
3- 2溫度控制系統介紹 35
3-3微量流速系統介紹 36
第四章 實驗設計與方法 37
4-1實驗設計流程圖 37
4-1-1 共軛焦顯微鏡觀測Actin細胞骨架於細胞三度空間內的分佈結構 37
4-1-2 QD-EGF與EGFR專一性結合測試以及利用EGF提高QD-EGF胞吞作用之實驗驗證 39
4-1-3 PD153035抑制EGFR活性的劑量評估以及PD153035抑制劑之可逆性驗證 41
4-2實驗材料與儀器設備 43
4-2-1 實驗材料 43
4-2-2儀器設備 44
4-3實驗方法 45
4-3-1細胞培養及處理 45
4-3-2細胞置入蓋坡片之處理 45
4-3-3 玻片微流道之製作 47
4-3-4量子點表面標定biotin-EGF之製備 47
4-3-5 Alexa 635染劑之配製 48
4-3-6 標靶藥物PD153035之製備 48
第五章 結果與討論 49
5-1利用共軛焦顯微鏡觀測Actin細胞骨架於細胞三度空間內的分佈結構 49
5-2 量子點與不同細胞株之專一性結合反應測試 52
5-3 增加EGF濃度藉以提高QD-EGF胞吞作用之實驗驗證 57
5-4 PD153035對細胞EGFR之抑制與再活化反應觀測 59
5-4-1 PD153035對細胞EGFR活性的抑制劑量與EGFR再活化反應驗證 59
5-5 QD-EGF於單一活細胞內部之運輸作用定量量測 65
第六章 結論與未來方向 69
參考文獻 72



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