臺灣博碩士論文加值系統

表皮生長因子受體 (EGFR)突變的非小細胞肺癌病人對EGFR酪氨酸激酶抑制劑(TKIs)反應良好，但病人最後皆產生抗藥性及復發。上皮細胞的上皮-間質轉化 (EMT) 可預測EGFR TKIs的抗藥性，但其分子機制迄今仍不清楚。本研究目標:探討細胞對gefitinib產生抗藥性時EMT調控因子的角色。方法: gefitinib敏感性細胞株(PC9)和gefitinib抗藥性細胞株(PC9/gef)用即時反轉錄聚合酶連鎖反應(RT-PCR)和西方墨點法測定EMT調控因子表現量﹔體外試驗方面，利用分子操控如過度或靜默表現等方法來研究EMT調控因子對gefitinib抗藥性的影響，體內試驗則採用活體腫瘤異種移植老鼠模式(xenograft mouse model)。另外，為了瞭解gefitinib抗藥性病人EMT調控因子mRNA的表現量，收集44個肺腺癌病人的惡性肋膜積液的癌細胞，培養後進行即時反轉錄聚合酶連鎖反應。結果: PC9/gef細胞的Slug 訊息核醣核酸和蛋白表現量明顯比PC9高，而其他調控者Snail, twist, Zeb-1 在這兩細胞株表現量類似，減弱Slug表現的PC9/gef細胞降低gefitinib抗藥性，反之，Slug過度的表現對gefitinib誘發PC9細胞凋亡提供了保護作用，減弱Slug表現的PC9/gef細胞主要透過增加Bim表現量和活化caspase 9來降低gefitinib抗藥性。Slug促進裸鼠異位移植腫瘤的生長，即使在gefitinib治療的期間。臨床檢體檢測顯示，在EGFR酪胺酸激酶抑制劑治療後比治療前，Slug 表現量顯著增加。結論，Slug促進gefitinib抗藥性產生而且未來可能成為治療肺癌、研發新藥的新標靶。

Rationale: Non-small cell lung cancers carrying epidermal growth factor receptor (EGFR) mutations respond well to EGFR tyrosine kinase inhibitors (TKIs), but patients ultimately develop drug resistance and relapse. Although epithelial-mesenchymal transition (EMT) can predict resistance to EGFR TKIs, the molecular mechanisms are still unknown.
Objectives: To examine the role of EMT regulators in resistance to gefitinib.
Methods: The expression level of EMT regulators in gefitinib-sensitive cells (PC9) and gefitinib-resistant cells (PC9/gef) was determined using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Molecular manipulations (silencing or overexpression) were performed to investigate the effects of EMT regulators on gefitinib resistance in vitro, and a xenograft mouse model was used for in vivo confirmation. In addition, cancer cells from fifty-four patients with malignant pleural effusions of lung adenocarcinoma were collected for analysis of EMT regulator mRNA by quantitative real-time RT-PCR.
Results: Slug expression, but not that of snail, twist or zeb-1, was significantly increased in PC9/gef compared to PC9 cells. Slug knockdown in PC9/gef cells reversed resistance to gefitinib, and overexpression of Slug in PC9 cells protected cells from gefitinib induced-apoptosis. Silencing of Slug in gefitinib-resistant cells restored gefitinib-induced apoptosis primarily through Bim upregulation and activation of caspase-9. Slug enhanced tumor growth in a xenograft mouse model, even with gefitinib treatment. In clinical samples, Slug expression was significantly higher in cancer cells with resistance to EGFR TKIs than in treatment-naïve cancer cells.
Conclusions: Slug contributes to the resistance to gefitinib and may be a potential therapeutic target for treating resistance to EGFR TKIs.

目錄
正文目錄....................................................................................................I
中文摘要..................................................................................................III
英文摘要...................................................................................................V
正 文 目 錄
第一章 前言 1
第一節 肺癌 2
第二節 表皮生長因子受體與肺癌的關係 2
第三節 表皮生長因子受體的分子標靶藥物敏感性相關因素 4
第四節 表皮生長因子受體之分子標靶藥物的抗藥性機制 6
第五節 上皮間質轉換調節因子Slug 9
第六節研究動機 10
第二章 材料與方法 12
第一節 材料 13
第二節 藥品與試劑: 13
第三節 方法 14
第三章 實驗結果 25
第一節 建立續發抗藥性體外模式 26
第二節 PC9/gef細胞對gefitinib抗藥性非因表皮生長因子受體T790M或c-MET擴增所造成。 27
第三節 PC9/gef細胞過度表現上皮間質轉化調節因子Slug。 28
第四節 核醣核酸干擾技術(RNA interference)減弱Slug表現 29
第五節 減弱Slug可逆轉PC9/gef細胞對gefitinib的抗藥性 30
第六節 Slug保護PC9細胞不產生gefitinib誘發的細胞凋亡。 31
第七節 Slug透過降低Bim表現來抑制gefitinib誘發的細胞凋亡 32
第八節 肺癌病患的Slug在表皮生長因子受體酪胺酸激酶抑制劑產生續發抗藥性的表現量增加 34
第四章 討論 35
第五章 表 41
第六章 圖 45
第七章 參考文獻 58

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