臺灣博碩士論文加值系統

Mac-2 binding protein (Mac-2 BP)是一個分子量大小約為90 kDa的分泌型醣蛋白，過去許多研究指出Mac-2 BP在多種癌症中高度表現，然而其詳細之分子機制與功能至今尚不明瞭。本實驗室先前的研究中發現Mac-2 BP在口腔癌細胞株與口腔癌病人檢體中過量表達，且抑制Mac-2 BP在口腔癌細胞株中之表現導致細胞移動能力下降，顯示Mac-2 BP在口腔癌移動能力中可能扮演重要角色。在本研究中，以Mac-2 BP穩定抑制表現之細胞株為研究模式，藉由質譜數據分析以及生物資訊之預測，大規模檢視其細胞內之蛋白質體與分泌蛋白體中之分子變化，以及可能遭受影響之訊息傳遞路徑。質譜數據以及後續驗證之結果皆顯示細胞外基質蛋白質fibronectin (FN)、laminin (LN)以及細胞膜表面受器integrin β1等細胞貼附移動相關之蛋白質表現量上升，過去文獻報導細胞外基質與整連蛋白質之訊息傳遞促進細胞貼附或是移動之能力上升，因此我們推測抑制Mac-2 BP表現可能改變了細胞貼附能力或是阻斷了細胞內外訊息傳遞路徑進而導致細胞遷移能力顯下降。免疫螢光染色與共軛焦顯微鏡技術觀察之結果發現穩定抑制Mac-2 BP之細胞中，黏著斑(focal adhesion)面積較控制組細胞大，然而細胞之貼附能力測試並無顯著差異。此外；穩定抑制Mac-2 BP之細胞對於FN刺激之敏感度似乎略低於控制組細胞，但是差異並不顯著。此兩項結果間接顯示Mac-2 BP可能影響了多重的途徑，而貼附能力的微小變化可能並非影響細胞移動能力顯著下降之主因。另一方面，在質譜數據分析中，我們發現細胞之分泌蛋白體在Mac-2 BP穩定抑制表現之細胞中變動劇烈，基因本體(Gene Ontoogy)預測結果指出部分分泌蛋白體中顯著變化之蛋白質與細胞中蛋白質運輸、分泌途徑相關。這項預測暗示著Mac-2 BP可能參與調控蛋白質運輸與分泌途徑，因而影響細胞內外訊息之平衡進而導致細胞遷移等功能的改變。

Mac-2 binding protein (Mac-2 BP) is a secreted glycoprotein of 90-100 kDa. Although elevated Mac-2 BP levels have been observed in tissues and sera of patients with various cancer types, the functions of Mac-2 BP remain largely unknown. Previous study from our lab revealed that Mac-2 BP is overexpressed in oral squamous cell carcinoma (OSCC) and could be highly secreted by OSCC cell lines. Furthermore, Mac-2 BP knockdown in OEC-M1 caused drastic inhibition of cell migration, suggesting that Mac-2 BP may play an important role in the mobility of OSCC cells. In this study, we investigate the global effect of Mac-2 BP knockdown in OC3 cells on the total cell proteome and the secreted proteome (secretome). Since previously researches have reported that ECM-integrin signaling pathway may improve cell mobility, these data imply that Mac-2 BP may participate in ECM-integrin signaling pathway, and stably knocked down Mac-2 BP alters cell mobility by regulating cell adhesion or blocking the signal transduction. Quantifying confocal microscope image of immunofluorescence staining indicated that the average size but not the number of focal adhesions was larger in Mac-2 BP knockdown (KD) cells than in control cells. However, we could not observe the difference in cell adhesion function. Moreover, FAK activation of Mac-2 BP knock down cells showed low sensitivity to FN stimulation, although the phenomenon is not significant. These results demonstrate that Mac-2 BP may affect multiple pathways, and the slightly change of adhesive ability is not the main factor for altering cell mobility. Additionally, the bioinformatics analysis show the dramatic change of secretome in Mac-2 BP KD cells, which may be caused by exceptional transportation and secretion. These results indicate that Mac-2 BP may in turn affect cell mobility by varying the signal equilibrium.

目錄
指導教授推薦書
口試委員會審定書
長庚大學碩博士論文授權書
誌謝 iv
Abstract vi
中文摘要 viii
目錄 x
圖目錄 xiv
表目錄 xvi
一、前言 1
1. 口腔癌 (Oral Squamous Cell Carcinoma；OSCC ) 1
2. Mac-2 Binding Protein (Mac-2 BP) 2
3. 細胞貼附與遷移 5
4. 細胞外基質蛋白質與連整蛋白質訊息傳遞對於細胞移動之影響 7
4.1. 整連蛋白質 (integrin) 7
4.2. 纖維粘連蛋白質 (Fibronectin；Fn) 9
4.3. 層粘連蛋白質(Laminin, LN) 11
5. 本論文研究動機 12
二、材料與方法 14
1. 藥品與材料 14
1.1. 一般藥品與材料 14
1.2. 抗體 14
2. 實驗方法 14
2.1. 細胞培養 14
2.2. 細胞移動能力測試 15
2.3. 細胞侵略能力測試 15
2.4. 質譜數據分析 16
2.5. 細胞內蛋白質萃取 17
2.6. 細胞分泌蛋白質萃取 17
2.7. 蛋白質定量分析 (BCA Assay) 18
2.8. 聚丙烯醯胺膠體電泳(SDS-PAGE) 18
2.9. 西方墨點法 (Western Blotting) 19
2.10. RNA 萃取 (RNA extraction) 20
2.11. 反轉錄聚合酶鏈鎖反應 (Reverse transcription polymerase chain reaction；RT-PCR) 21
2.12. 定量即時聚合酶鏈鎖反應 (Real-time polymerase xiichain reaction；quantitative polymerase chain reaction；Q-PCR) 21
2.13. 細胞表面 ITGB1 表現量偵測 22
2.14. 免疫螢光染色 (immunofluorescence) 22
2.15. FN 刺激 ITGB1 訊息傳遞 23
三、實驗結果 24
1. Mac-2 BP Knockdown OSCC 導致口腔癌細胞移動與侵略能力下降 24
2. Mac-2 BP Knockdown OSCC 之蛋白質體及分泌體數據分析與相關訊息傳遞途徑預測 25
2.1. 質譜數據初步篩選 25
2.2. 利用 MetaCore pathway analysis 預測相關之訊息傳遞途徑 29
2.3. 利用 STRING 分析顯著變化蛋白質之基因本體 (Gene Ontology) 31
2.4. 利用 signalP 預測蛋白質運輸之訊號序列 36
2.5. ExoCarta 資料庫比對 38
3. 細胞外基質蛋白質 Fibronectin 與 Laminin 之驗證 39
3.1.纖維粘連蛋白質 (Fibronectin；FN)之 mRNA 與蛋白質表現量在 Mac-2 BP knockdown 之 OSCC 皆增加 39
3.2. 層粘連蛋白質 (Laminin；Ln) 蛋白質 laminin 5 與laminin β1/γ1 表現量在 Mac-2 BP 穩定抑制表達之細胞株中顯著增加 40
4. 細胞表面穿膜受器整連蛋白質 (integrin β1；ITGB1)於膜上表現量顯著上升 40
5. Mac-2 BP knockdown 之細胞中 Focal adhesion 面積大於控制組細胞 41
6. FN 刺激整連蛋白之訊息傳遞 42
四、討論 45
參考文獻 54

圖目錄
圖一、在口腔癌細胞中抑制Mac-2 BP表現導致細胞移動與侵略能力下降 xiv
圖二、質譜身分鑑定數據之初步篩選流程 xvi
圖三、蛋白質含量分布圖 xviii
圖四、資料整合流程圖 xix
圖五、MetaCoreTM Pathway analysis資料示意圖 xx
圖六、MetaCore Pathway Analysis分析結果 – CE與CM交集之449個蛋白質可能參與的訊息傳遞途徑前十名 xxi
圖七、MetaCore Pathway Analysis分析結果 – 85個顯著變化之蛋白質可能參與之訊息傳遞途徑前十名 xxii
圖八、Cell adhesion_Role of tetraspanins in the integrin-mediated cell adhesion xxiii
圖九、分泌蛋白體之蛋白質分群方法 xxiv
圖十、CM上升且CE上升之蛋白質Gene Ontology分析。 xxv
圖十一、CM上升CE不變之蛋白質Gene Ontology分析。 xxvi
圖十二、CM上升CE未鑑定到之蛋白質Gene Ontology分析。 xxvii
圖十三、CM下降CE上升之蛋白質Gene Ontology分析。 xxviii
圖十四、CM下降CE不變之蛋白質Gene Ontology分析。 xxix
圖十五、CM下降且CE下降之蛋白質Gene Ontology分析。 xxx
圖十六、CM下降CE未鑑定到之蛋白質Gene Ontology分析。 xxxi
圖十七、Mac-2 BP knockdown 細胞中Fibronectin之mRNA與蛋白質表現量顯著上升 xxxii
圖十八、laminin 5與laminin β1/γ1在Mac-2 BP穩定抑制表達之細胞中顯著增加 xxxiii
圖十九、integrin β1在Mac-2 BP穩定抑制表達之細胞表面高度表現 xxxv
圖二十、Mac-2 BP knockdown細胞中之黏著斑 (Focal adhesion)面積大於控制組細胞 xxxvi
圖二十一、FN 刺激連整蛋白質下游訊息傳遞之結果 xxxvii

表目錄
表一、Role of tetraspanins in the integrin-mediated cell adhesion訊息傳遞途徑中顯著變化之蛋白質比例與其所對應之基因名 xxxix
表二、SignalP預測結果 xl
表三、ExoCarta 蛋白質比對結果 xli
附表一、 一般藥品與材料 xlii
附表二、抗體 xliii
附表三、 Mac-2 BP knockdown OSCC之細胞萃取液蛋白質體與分泌蛋白體中皆顯著上升之蛋白 xliv
附表四、Mac-2 BP knockdown OSCC之細胞萃取液蛋白質體與分泌蛋白體中皆顯著下降之蛋白質 xlv

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