臺灣博碩士論文加值系統

Galectin-1(Gal-1)屬β-galactoside-binding animal lectins家族的一員，已知參與腫瘤細胞的轉型、附著、遷移及避開免疫攻擊等反應，Gal-1在口腔癌前期會大量表現在腫瘤附近的間質組織(tumor-associated stroma)及侵犯前緣與轉移至淋巴結的腫瘤細胞，並與前期口腔癌的預後有關。基於這些和腫瘤生物息息相關的反應，本研究利用基因沉默(gene silencing)的方法去探討細胞裡內生性Gal-1對於口腔癌的發展過程及其生物活性，發現抑制細胞體內的Gal-1會促進口腔癌細胞的生長、增加細胞週期S期的量、抑制細胞的遷移、及降低MMP-2及MMP-9蛋白活性的表現；且降低Gal-1的表現量可減少small GTPase cdc42的活化而明顯改變細胞表面filopodia的形態與數量進而抑制細胞的遷移。接著利用Tandem affinity purification (TAP)純化策略與質譜學(mass spectrometry, MS)的方法找出和Gal-1有交互作用的蛋白質Disheveled-associated activator of morphogenesis 1 (Daam1)和histone H4，雖然免疫共沉澱法(Coimmunoprecipitation)及哺乳動物雙雜交系統(mammalian two-hybrid)未能證明Gal-1和histone H4及Daam1有交互作用，但我們的結果卻發現到Gal-1和Daam1蛋白在細胞內的表現量是有正相關，且免疫螢光染色法(Immunocytometry)也指出Gal-1及Daam1在細胞內有colocalized，顯示Gal-1和Daam1之間的互動和口腔癌的發展過程有關聯，釐清細胞內Gal-1和Daam1的蛋白質聯絡網絡將有助於定位Gal-1於口腔癌細胞的增生及細胞遷移所扮演的角色。

Galectin-1(Gal-1) is a β-galactoside-binding lectin and involved in multiple biological functions, such as cell adhesion, proliferation, migration, apoptosis, inflammation, tumor progression and metastasis. Our previous investigations have shown that Gal-1 was overexpressed in the tumor-associated stroma as well as the invasion front during early oral carcinogenesis and correlated with worse prognosis of oral cancer. In this study we used siRNA of Gal-1 to determine the function of intracellular Gal-1. We found that silencing Gal-1 stimulates proliferation, increases cell cycle S phase, inhibits cell migration, and decreases the expression of MMP-2 and MMP-9. The migration inhibition of silencing Gal-1 might be due to the inhibition of small GTPase cdc42 activity and cytoskeleton rearrangement and significant decrease in the length and number of filopodia. Next, using a tandem affinity purification (TAP) method to purify the protein complex of Gal-1 and subsequent mass spectrometry to identify the associated proteins. Disheveled-associated activator of morphogenesis 1 (Daam1) and Histone H4 proteins were first identified as Gal-1 associated proteins by such approaches. Co-immunoprecipitation and mammalian two-hybrid analysis did not demonstrate that Gal-1 interacted with histone H4 and Daam1. However, Gal-1 was strongly sub-cellular colocalized with Daam1, and the expression of Daam1 was significantly decreased while gal-1 was silencing. These results implied that Daam1 might have a role in Gal-1-regulated cell functions. To further clarify the interacting network of Gal-1 and Daam1 might help to gain insight into the Gal-1 functions in oral carcinogenesis.

中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
表目錄 VII
圖目錄 VIII
緒論 1
一、口腔癌 (Oral cancer) 2
二、Galectin-1蛋白 3
三、核糖核酸干擾現象(RNA interference, RNAi) 5
四、Tandem affinity purification (TAP)純化策略 6
研究動機 8
材料與方法 10
解凍細胞 11
細胞繼代培養 12
細胞計數 13
細胞保存 13
RNA干擾作用轉染 (RNA interference transfection) 14
細胞蛋白樣本製備 15
蛋白質定量 16
蛋白質電泳 (SDS polyacrylamide gel electrophoresis) 16
西方墨點法 (Western blot) 18
細胞增生分析 (Cell proliferation assay) 20
細胞週期分析及量化 21
細胞移動分析 (Cell migration assay) 22
細胞F-actin螢光染色 24
Small GTPase pull down試驗 25
Zymography分析 26
哺乳動物細胞轉染作用 27
TAP純化 29
On- beads digestion 31
SYPRO Ruby染色法 32
免疫螢光細胞染色 33
聚合酵素連鎖反應 (Polymerase chain reaction, PCR) 34
pCMX-GAL4-H4質體構築 35
哺乳動物雙雜交 38
細胞核粹取液粹取法 39
免疫共沈澱法(co-immunoprecipitation, co- IP) 40
實驗結果 42
1. Gal-1 siRNA有效降低人類口腔癌細胞株HSC-3內Gal-1蛋白的表現 43
2. 抑制Gal-1蛋白的表現促進口腔癌細胞HSC-3的增生 43
3. 抑制Gal-1蛋白的表現對細胞週期的影響 43
4. 抑制Gal-1蛋白的表現抑制口腔癌細胞HSC-3的遷移 44
5. 抑制Gal-1蛋白的表現改變口腔癌細胞HSC-3細胞表面filopodia的形態及數量 44
6. 抑制Gal-1蛋白的表現量可減少small GTPase cdc42的活化 45
7. 抑制Gal-1蛋白的表現降低口腔癌細胞HSC-3裡MMP-2及MMP-9蛋白的表現 46
8. 利用TAP純化策略找尋和Gal-1有交互作用的蛋白 46
8.1 TAP純化流程產物確認 --- streptavidin resin 47
8.2 TAP純化流程產物確認 --- calmodulin resin 48
9. 利用SDS-PAGE檢測純化後的Gal-1蛋白複合體 48
10. On beads digestion及質譜分析的結果 49
11. Gal-1和Daam1在細胞內有colocalization 49
12. 抑制Gal-1蛋白Daam1蛋白的表現量也隨之下降 50
13. 免疫共沈澱法(co-immunoprecipitation, co-IP)無法證明Gal-1和Daam1有交互作用 50
14. 免疫共沈澱法(co-IP)無法證明Gal-1和histone H4有交互作 51
15. 哺乳動物雙雜交系統(mammalian two-hybrid)無法證明 Gal-1和histone H4有交互作用 51
討論 53
結論 63
參考文獻 64
附錄一、儀器設備 86
附錄二、英文縮寫 87

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