癌症是國人十大死因之首，在男性癌症中口腔癌排名第四。而在癌症發生率來看，91 年到 96 年，十大癌症中以口腔癌增加幅度最高(3.9%)，推測主要是台灣地區特有的飲食文化而造成。癌症高致死率為癌症的轉移，在腫瘤細胞轉移的過程中，細胞型態會改變稱 EMT(Epithelial to Mesenchymal transition)機制，此機制參與了癌細胞侵襲轉移。目前研究發現，Axl 會大量表現在腎臟癌、大腸癌、胃癌、皮膚癌、骨癌等的腫瘤組織上。而 Axl 為 RTK (Receptor Tyrosine Kinase)家族成員之一，透過與其 ligand Gas6 (Growth arrest-specific gene 6)的binding 活化下游的訊息傳遞路徑 (PI3K、MAPK) ，進而促進癌細胞轉移，但其在口腔癌中分子機轉尚未明確。因此本研究將探討 Axl 在口腔癌上的所扮演的角色與調控的機制。材料與方法:使用口腔鱗狀上皮癌細胞株 YD38，利用 lentiviral 建立 Axl knockdown 的細胞株(shAxl574、shAxl575) ，再觀察其 migration、invasion、colony-formation、proliferation ，並且利用螢光免疫、流式細胞儀及西方點墨法來觀察細胞週期變化以及其他 EMT 相關的蛋白質表現量 (E/P/N-cadherin、β-catherin、Snail/Slug) 。結果: 與 Parental cell 相較 Axl knockdown 的細胞株型態上明顯變得比較聚集(epithelial like)，在細胞週期 G1 停滯的比例也都有顯著提升現象，同時在subG1 的比例也相對提升，代表著有 apoptosis 的情形發生。在 Axl knockdown 的細胞株其運動性、侵襲性和存活率也較 parental cell 低。在 EMT 蛋白質的表現上 (如mesenchymal 的指標 P/N-cadherin、β-catherin、Snail/Slug) Axl
knockdown 的細胞株(shAxl574、shAxl575)表現皆降低，顯見 Axl本身對口腔癌細胞惡化與 EMT 變化的重要性。結論:研究結果闡明 Axl的確會參與口腔癌侵襲能力及 EMT 變化，並調控轉移所引發的相關分子機制，進而說明了 Axl 在口腔癌細胞惡化過程中所扮演的角色。

Cancer has ranked the number one cause of death in Taiwan. Oral cancer rank fourth of male’s cancer , which has the highest rate of increase . Cancer metastasis to distant site is the most common cause of
death of patient. This malignant progression is enhanced by tumor cell activation of an epithelial to mesenchymal transition (EMT). Axl is a member of the TAM receptor tyrosine kinase (RTK) that share the vitamin K-dependent ligand Gas6 (Growth arrest specific 6). TAM family receptor regulate a diverse range of cellular responses. Axl activation is linked to several signal transduction pathway, including PI3K/Akt, MAP kinase and others. However, the role of Axl in oral cancer progression remains
unknown.
In this study, we test the hypothesis that Axl is a critical factor for oral cancer metastasis and regulator of epithelial to mesenchymal transition. Here we show that Axl expression is required to maintain the mesenchymal-like invasiveness of oral cancer cell. Inhibition of Axl expression attenuates oral cancer cell migration, invasion, and matrix metalloproteinase activity. We demonstrate that EMT factors were induced by Axl modulation in oral tumor. Axl knockdown resulted in G1 phase arrest and apoptosis. These findings suggest that Axl participated in the process of EMT that is required for oral cancer metastasis. In conclusion , our data show that Axl contributes to EMT and regulates cell functions in oral cancer . Moreover , these results provide a molecular explantation for oral cancer cell migration during EMT by Axl as a key critical factor in this process .

謝誌........................................Ⅰ
目錄........................................Ⅱ
圖目錄......................................Ⅳ
縮寫表......................................Ⅵ
中文摘要.....................................Ⅶ
英文摘要.....................................Ⅸ
第一章 緒論...............................01

第一節 口腔癌..............................01
第二節 Axl接受器...........................02
第三節 Gas6 結合子.........................05
第四節 訊息調控路徑.........................06
第五節 EMT機制.............................07
第六節 EMT Marks..........................09

第二章 實驗材料與方法.........................13

第一節 實驗材料.............................13
第二節 實驗方法.............................15

第三章 結果.................................37

第一節 利用西方墨點法來觀察口腔癌細胞株的致癌基因 Axl的表現................................37
第二節 利用慢病毒（lentivirus）系統降低口腔癌細胞 YD38 細胞株Axl mRNA表現量................37
第三節 Axl 的表現調控著口腔癌細胞的移動性和侵襲性........................................38
第四節 Axl 調控口腔癌細胞的群落生成速度與 MMP2/9 活性.....................................40
第五節 Axl 調控口腔癌細胞中 EMT 轉錄因子...............................................40
第六節 Axl 調控口腔癌細胞的生長與細胞週期.................................................42
第七節 Axl 透過 PI3K/AKT 路徑來調控口腔癌細胞............................................44

第四章 討論............................................................................46

第一節 選用 YD38 口腔癌細胞作為實驗模型的原因..............................................46
第二節 Axl 誘導口腔癌細胞的移動性和侵襲性的發生............................................46
第三節 MMP2/9 會促使口腔癌細胞轉移能力發生................................................47
第四節 口腔癌上 Axl 影響 EMT 轉錄因子的表現................................................48
第五節 Axl 不影響 β-catenin 轉錄因子入核的表現............................................48
第六節 Axl 調控細胞週期與細胞凋亡.........................................................50
第七節 Axl 在癌症治療上扮演的角色..........................................................51

第五章 結論.............................................................................52
圖表.......................................................................................53
參考文獻...................................................................................75

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