臺灣博碩士論文加值系統

自民國九十年起肺癌成為國人十大惡性腫瘤之首，其誘發原因尚未明瞭，推測與空氣品質、廚房油煙、吸菸或基因有關，且肺癌細胞遷移能力強，可經由移動、附著、侵入等步驟遷移至其它器官造成更大傷害且不易進行治療和控制。已有研究與文獻指出罹患肺腺癌且預後效果較差的病患，其肺腺癌細胞中含有大量岩藻醣轉移酶 4 (Fucosyltransferases 4, FUT4)。先前研究指出將 fut4 基因轉殖入肺腺癌細胞株 A549 (A549 FUT4) 中，並透過蛋白質體學技術發現 fut4 可誘使熱休克蛋白 (Heat shock protein 27, HSP 27)、半乳糖凝集素-1 (Galectin-1) 與過氧化氧化還原酶-1 (Peroxiredoxin-1, PRDX-1) 三種蛋白質大量表現。回顧過去的文獻發現 Galectin-1 在癌細胞上扮演重要角色，尤其是它使癌細胞的侵入及移動能力增加這部分特別值得關注。故選定 Galectin-1 作為本篇研究主題。本篇研究首先藉由西方點墨法 (Western Blot assays) 和即時定量聚合酶鏈鎖反應 (Real-Time Quantitative Polymerase Chain Reaction, RT-qPCR) 驗證後發現 A549 FUT4 中 Galectin-1 蛋白質和 mRNA 表現量高於 A549 Mock (不含 fut4 基因的肺腺癌細胞株)，而後透過傷口癒合試驗 (Wound healing assays) 與 Transwell 試驗發現 A549 FUT4 平行、垂直移動能力皆較A549 Mock 差，而在 Transwell (含 matrigel) 試驗中則發現 A549 FUT4 侵入能力較 A549 Mock 強。此外，透過 RT-qPCR 發現 A549 FUT4 中基質金屬蛋白酶 2 (Matrix metalloproteinases 2, MMP-2)、基質金屬蛋白酶 9 (Matrix metalloproteinases 9, MMP-9) mRNA 表現量皆高於 A549 Mock。實驗中進一步利用 RNA 干擾技術 (RNA interference, RNAi) 將 A549 FUT4 中的 Galectin-1 默化 (knockdown) 使蛋白質表現量下降，以探討 A549 FUT4 的侵入、移動能力是否與 Galectin-1 有關，並透過 Western Blot assays 驗證 RNAi 可有效降低 Galectin-1 表現量，並接近完全抑制。利用上述相同試驗觀察 knockdown Galectin-1 後的 A549 FUT4 移動、侵入能力與 MMP-2、MMP-9 mRNA 表現量，可以發現 knockdown Galectin-1 後 A549 FUT4 侵入、移動能力皆降低，且 MMP-2、MMP-9 mRNA 表現量皆被抑制，而由結果證實了 Galectin-1 與肺腺癌細胞移動與侵入能力之調控有關。

High expression of Fucosyltransferase 4 (FUT4) level was correlated to poor prognosis in lung cancer patients. In previous study, using proteomic technology showed Heat shock protein 27 (HSP27), Galectin-1 (Gal-1) and Peroxiredoxin-1 (PRDX-1) were significantly increased after fut4 gene was transfected into lung adenocarcinoma cell line A549 (A549 FUT4). Gal-1 plays an important role in cancer, especially in invasion and migration abilities. In this study, Gal-1 expression level was higher in A549 FUT4 than A549 Mock by Western Blot and RT-qPCR assays. The parallel and vertical migraton abilities of A549 FUT4 were lower than A549 Mock by Wound healing and Transwell assays, respectively However, using Transwell assays (with coating matrigel) to study invasion ability of A549 FUT4 and A549 Mock can find A549 FUT4 possessed better invasion ability. Expression of MMP-2, MMP-9 mRNA in A549 FUT4 were higher than A549 Mock by RT-qPCR assays. Furthermore, knockdown Gal-1 level in A549 FUT4 by RNA interference (RNAi) to study whether invasion and migration ability of A549 FUT4 is associated with expression of Gal-1. Through Western blot assays to validate effectiveness of RNAi that could find Gal-1 was effectively inhibited until 120 hours. Using the same assays to study invasion, migration abilities and expression of MMP-2, MMP-9 mRNA of A549 FUT4 after knockdown Gal-1. As a result, invasion and migration abilities of A549 FUT4 was decreased due to Gal-1 knockdown. The studies confirmed that invasion and migration abilities of lung cancer cells are positively associated with Gal-1.

縮寫表 1
摘要 2
Abstract 4
壹、研究目的及架構 6
貳、文獻整理 8
一、肺癌 8
二、半乳糖凝集素 (Galectins) 9
(一)、半乳糖凝集素 9
(二)、半乳糖凝集素-1 (Galectin-1) 10
三、細胞外基質對癌細胞之的影響 14
四、醣基 15
(一)、單醣的種類 15
(二)、醣蛋白的結構與功能 15
五、醣化 (Glycosylation) 15
六、岩藻醣轉移酶 (Fucosyltransferases) 17
(一)、岩藻醣與岩藻醣化 17
(二)、岩藻醣轉移酶 17
(三)、岩藻醣轉移酶分類 18
(四)、岩藻醣基生成路徑 19
參、研究內容 21
第一章 A549 FUT4 與 A549 Mock 的生長、侵入和移動能力 22
一、前言 22
二、材料與方法 22
三、結果 46
四、討論 49
五、圖表 52
第二章 Knockdown Galectin-1 以探討 A549 FUT4 中 Galectin-1 表現量是否與侵入、移動能力有關 64
一、前言 64
二、材料與方法 64
三、結果 73
四、討論 75
五、圖表 78
肆、結論 87
伍、參考文獻 88
陸、謝辭 100

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