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系統識別號 U0026-0408201012105800
論文名稱(中文) α-catulin在癌細胞侵襲所扮演的角色
論文名稱(英文) Role of α-catulin in cancer invasion
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 98
學期 2
出版年 99
研究生(中文) 邱思螢
研究生(英文) Szu-Ying Chiu
學號 t4697401
學位類別 碩士
語文別 中文
論文頁數 77頁
口試委員 指導教授-陳玉玲
口試委員-洪澤民
口試委員-袁國
口試委員-鄭宏祺
中文關鍵字 α-catulin  整合素αvβ3  NF-κB  纖連蛋白  細胞爬行能力 
英文關鍵字 α-catulin  Integrin αvβ3  NF-κB  Fibronectin  Migration  Invasion 
學科別分類
中文摘要 從1982年開始癌症已經居為國人十大死因首位,而近年口腔癌已為國人男性癌症死亡之第四位。α-catulin(α-catenin-related protein)為α-catenin相關蛋白,其大小為82kDa。在先前的研究指出α-catulin類似catenin和vinculin家族,此蛋白最早是在誘發胰臟癌細胞分化和凋亡時所發現表現量較少的蛋白,並發現參與Rho的傳訊路徑,和細胞貼附、細胞移行有關,以及可以透過和IKK-β結合去調控NF-κB去使細胞抵抗凋亡,但α-catulin的細胞功能及其在腫瘤發展的角色尚未清楚。先前實驗室發現抑制口腔癌細胞株(OC2)及肺癌細胞株(A549)中α-catulin的表現,發現細胞增生情況下降且細胞有老化的現象。利用Real-time PCR分析臨床檢體發現口腔癌組織的α-catulin表現量高於正常組織。因此本實驗目的想探討α-catulin大量表現在癌細胞中所造成的影響。初步在肺癌細胞株(A549)中發現,α-catulin分佈於細胞核及細胞質。我們也在α-catulin大量表現的細胞可以發現絲狀偽足(Filopodia)的密度和長度有增加的情況。接著進一步探討α-catulin對細胞的增生、爬行能力、侵襲能力及貼附能力的影響,結果發現大量表現α-catulin對細胞的增生沒有影響,但卻會增加細胞的爬行能力、侵襲能力及貼附能力。為了更進一步去探討α-catulin參與在細胞爬行能力、侵襲能力的機制,我們也發現大量表現α-catulin會使纖連蛋白(Fibronectin)及相關的整合素(Integrin)α5β1、αvβ3表現量上升,但抑制α-catulin卻只發現纖連蛋白(Fibronectin)及相關的整合素(Integrin)αvβ3表現量下降。利用整合素(Integrin)αvβ3中和抗體以及NF-κB 的抑制劑(Bay-117082),發現均可抑制細胞α-catulin所誘導的細胞移行作用,並且也發現處理Bay-117082可以抑制細胞整合素(Integrin) β3的表現量下降。綜合以上結果顯示,α-catulin可能是透過調控NF-κB而影響纖連蛋白(Fibronectin)及相關整合素(Integrin)αvβ3所參與的細胞移行機制。
英文摘要 Cancer has been the leading cause of death in Taiwan since 1982. Currently, oral cancer is the fourth cause of death for male cancer patients. α-Catulin is an 82-kDa α-catenin-related protein containing regions of strongly homology with α-catenin and vinculin. It has been known to be down-regulated in pancreatic cancer cells undergoing differentiation and apoptosis, modulated the Rho pathway signaling, and thus may involve in cell adhesion and cell migration. It has been also known to regulate NF-κB through binding IKK-β and confer resistance to apoptosis, but the role of α-catulin in cancer is unclear. In our previous study, we found that knockdown of α-catulin gene in OC2 and A549 dramatically decreased cell proliferation and contributed to cellular senescence. The mRNA levels of α-Catulin were significant increased in clinical oral squamous cell carcinoma (OSCC) tissues. In the present study, we seek to clarify its functional role in cancer progression by increasing α-catulin expression in cancer cells. We found that the subcellular distribution of α-catulin in A549 lung cancer cells is located in both nucleus and cytoplasm. α-Catulin overexpression increased the filopodia density and length of A549 cells. α-Catulin overexpression doesn’t influence cell proliferation but significantly enhances cancer cell migration, adhesion, and invasion. To dissect the underlying mechanism of α-catulin-promoted cancer cell migration and invasion, we found that α-catulin increases the expression of fibronectin and fibronectin-associated integrin α5β1 and αvβ3. Contrarily, α-catulin knockdown only decreases the expression of fibronectin and αvβ3. We also found that NF-κB inhibitor, Bay117082, decreases the expression of integrin β3 and suppresses α-catulin-promoted cell migration. Blocking αvβ3 signaling pathway by using neutralizing antibody effectively inhibits α-catulin-promoted cell migration. Taken together, our present study revealed that α-catulin might through regulating NF-κB activity to increase the expression of fibronectin and αvβ3, and then promote cell migration
論文目次 目 錄
中 文 摘 要 I
英 文 摘 要III
致 謝 V
目 錄 VII
表目錄 X
圖目錄 XI
附圖目錄 XIII
附錄目錄 XIV
英文縮寫檢索表 XV
緒 論 1
癌症的轉移 1
α-catulin 2
整合素(Integrin)在癌細胞中所扮演的角色 3
小GTPase Rho 家族蛋白質(small GTPase Rho protein family),在癌細胞中所扮演的角色 4
轉錄因子NF-κB (Nuclear factor-κB) 在癌細胞中所扮演的角色 5
研究動機 8
材料與方法 9
一、癌細胞株(A549、CL-1-0、CL-1-5)培養 9
1-1 繼代培養 9
1-2 冷凍保存細胞 9
1-3 細胞解凍 10
1-4 細胞計數 10
二、細胞內RNA表現分析 11
2-1 RNA 萃取 11
2-2 RNA 及DNA的定量 12
2-3 反轉錄酶反應(Reverse transcription)12
2-4 聚合酶連鎖反應(polymerase chain reaction,PCR) 13
2-5 洋菜膠體電泳分析(Agarose gel electrophoresis)16
2-6 即時定量PCR (qRT-PCR)17
三、細胞蛋白質表現分析 18
3-1 細胞蛋白質收集 18
3-2 蛋白質定量 18
3-3西方墨點法(Western blot assay)19
3-3-1 蛋白質樣品之準備 19
3-3-2 SDS-聚丙烯胺膠體電泳 19
3-3-3 轉印(Electrotransfer)21
3-3-4 免疫轉漬法(Immunoblotting)21
四、GFP-α-catulin載體構築 22
五、GFP-α-catulin載體轉型作用(Transformation)23
六、GFP-α-catulin質體之細胞轉染(Transfection) 24
七、細胞螢光染色(Immunofluorescence)25
八、Lentiviral病毒質體的製備 26
8-1 過度表現病毒系統的製備(Overexpression system)26
8-2 抑制表現病毒系統的製備(shRNA system)27
九、α-catulin對肺癌細胞功能影響之實驗 28
9-1細胞增生分析(Proliferation assay)28
9-2 細胞貼附實驗(Adhesion assay) 29
9-4 細胞移行分析(Migration assay) 29
9-4-1 細胞移行抑制分析(Neutralization antibody) 30
9-5細胞侵襲分析(Invasion assay)31
十、細胞表面接受體分析-流式細胞儀(Flow cytometry)32
實驗 結 果 33
一、α-catulin在肺癌細胞株(A549、CL-1-0) 並不會影響細胞的生長 33
二、α-catulin會增加肺癌細胞株(A549、CL1-0)的侵襲能力 33
三、α-catulin會增加肺癌細胞株(A549、CL1-0)的移行 34
四、α-catulin會增加肺癌細胞株(A549)的貼附能力,特別是促進癌細胞對細胞外基質的纖連蛋白(Fibronectin) 的貼附能力 34
五、α-catulin主要表現在細胞質少量表現在細胞核 35
六、α-catulin影響A549細胞F-actin分布形態,並且增加細胞表面Filopodia的數目及長度 35
七、α-catulin影響肺癌細胞株(A549、CL-1-0)內整合素(Integrin αvβ3)的表現量 36
八、α-catulin透過增加Integrin αvβ3的表現促進細胞的移行能力 36
九、α-catulin透過整合素(Integrin) αvβ3所增加細胞的移行能力,NF-κB也可能參與其中 37
討 論 38
結 論 45
參考資料 46
自 述 77
表目錄
表一、α-catulin 表現與63組OSCC 臨床病理多因素之關係 70
圖目錄
圖一、α-catulin在各種不同細胞間的表現 55
圖二、過度表現α-catulin在肺癌細胞株(A549、CL-1-0) 並不會影響細胞的生長 56
圖三、過度表現α-catulin會增加肺癌細胞株(A549、CL1-0)的侵襲能力 57
圖四、過度表現α-catulin會增加肺癌細胞株(A549、CL1-0)的移行 58
圖五、過度表現α-catulin會增加肺癌細胞株(A549)的貼附於Fibronectin的能力 59
圖六、α-catulin主要表現在細胞質少量表現在細胞核 60
圖七、α-catulin影響A549細胞表面上F-actin分布形態,並且增加細胞表面Filopodia的數目及長度 61
圖八、過度表現α-catulin會增加肺癌細胞株(A549、CL-1-0)內纖連蛋白(Fibronectin)及相關的整合素(Integrin)α5β1、αvβ3 RNA表現量上升 62
圖九、抑制α-catulin會減少肺癌細胞株(CL-1-5)內纖連蛋白(Fibronectin)及相關的整合素(integrin)αvβ3 RNA表現量 63
圖十、過度表現α-catulin會增加肺癌細胞株(A549、CL-1-0)內纖連蛋白(Fibronectin)及相關的整合素(Integrin)β3蛋白質的表現量以及增加細胞表面Integrin αV表現量64
圖十一、抑制α-catulin會減少肺癌細胞株(CL-1-5)內纖連蛋白(Fibronectin)的表現量 65
圖十二、αvβ3中和抗體能夠有效的抑制由α-catulin所增加肺癌細胞株(CL-1-0)的移行能力 66
圖十三、NF-κB抑制劑(Bay11-7082)能夠有效抑制α-catulin所增加的整合素(Integrin)αvβ3的RNA表現量 67
圖十四、NF-κB抑制劑(Bay11-7082)能夠有效抑制α-catulin所增加的肺癌細胞株(CL-1-0)移行能力 68
圖十五 α-catulin在癌細胞轉移中可能參與的訊息傳遞機制 69
附圖目錄
附圖一、α-catulin在63組OSCC檢體中的表現 71
附圖二、減少α-catulin的表現,會使細胞有老化的現象 72
附圖三、α-catulin在轉移至淋巴結的OSCC檢體中表現較高 73
附錄目錄
附錄一:抗體 74
附錄二:儀器 75
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