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系統識別號 U0026-0812200911525776
論文名稱(中文) Galectin-1能夠增加口腔癌細胞增生、遷移、和貼附,並且促進口腔癌的進展
論文名稱(英文) Galectin-1 enhances OSCC cells proliferation, migration, and adhesion in vitro and promotes cancer progression in vivo
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 94
學期 2
出版年 95
研究生(中文) 蕭佳彥
研究生(英文) Chia-Yen Hsiao
電子信箱 usual-1@yahoo.com.tw
學號 t4693401
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-袁國
召集委員-賴明德
口試委員-吳梨華
中文關鍵字 遷移  細胞增生  貼附 
英文關鍵字 ERK  adherence  galectin-1  proliferation  migration 
學科別分類
中文摘要 口腔癌的發生率及死亡率為國人頭頸部癌症的第一位,近年來已成為男性國人惡性腫瘤的第四位,其平均死亡年齡比其他癌症早10年以上,這些年齡層的男性正是家庭經濟的主要支柱,是社會進步的主力。口腔癌死亡率居高不下的原因常是因為延誤診斷與治療,因此找到口腔癌早期診斷與治療的分子標的是當務之急。先前的文獻使用蛋白質體學比對口腔癌症組織與正常組織中蛋白質表現的差異,並指出了幾個具有高度發展性的蛋白質,包括heat shock protein 60 (HSP60), HSP27, B-crystalline, ATP synthase , calgranulin B, myosin, tropomyosin和galectin-1,他們都在口腔癌組織的細胞中被大量的表現,因此我們就選定了galectin-1作為本篇研究的主題。Galectins是一群可與-galactoside binding的lectins蛋白質家族,由許多種不同的形式的結構組成不同的galectin,他們具有兩種特性:1. 對-galactosides有鍵結活性,2. 具有相似的carbohydrate recognition domain (CRD)的序列。而他們在動物體內的功能雖然還不能被完全的瞭解,但是已經有證據指出這些lectins對於生長調節、細胞貼附、細胞移動、腫瘤形成、以及免疫系統的調節中扮演一個重要的角色。本實驗室先前的研究即指出,在口腔癌前期,以免疫組織染色法去偵測腫瘤組織之galectin-1的表現時,發現galectin-1大量表現在腫瘤的間質組織(stroma),尤其在癌症早期的表現特別明顯,顯示galectin-1在癌症前期的細胞周圍可能對於腫瘤的進程及其本身的生長具有一定程度的影響。腫瘤的微環境(tumor microenvironment)對整個癌化過程扮演重要的角色,因此本實驗的目的是要探討在口腔癌前期,細胞周圍的galectin-1大量增加,對腫瘤的生長能力造成怎樣的影響。Galectin-1首先被建構成pRSET-galectin-1並在大腸桿菌BL-21菌株大量表現,經由兩步驟的純化流程我們得到galectin-1的重組蛋白質,利用SDS-PAGE、Q-TOF確定其純度及分子量,便利用此galectin-1的重組蛋白質開始進行口腔癌細胞相關活性的研究。結果發現外加galectin-1蛋白質能促使口腔癌細胞的遷移、增生、及貼附,並隨著濃度的提升而增加其效果。探討其細胞訊息傳遞的途徑,發現galectin-1蛋白質促進細胞的增生可能與ERK的磷酸化是有相關性的。為了探討galectin-1蛋白質對口腔癌細胞活性影響的分子機轉,我們進一步進行致癌基因及激酶基因微陣列(oncogene/kinase chips)分析來了解galectin-1蛋白質對口腔癌細胞致癌相關基因表現的調控,並以RT-PCR及西方點墨法來確認基因晶片的正確性。結果發現AKIP(Aurora-A kinase interacting protein)、BRCA1(breast cancer susceptibility gene 1)及cyclin A1之mRNA的表現的確是被galectin-1蛋白質所調控,且BRCA1之蛋白質亦有減少的現象。由於這些基因都是跟細胞週期的調控有關係的,所以我們進行細胞週期的實驗,以流式細胞儀及PI染色來分析以galectin-1蛋白質處理細胞之後其細胞週期的變化。結果發現以galectin-1蛋白質處理過的細胞,其S期的細胞數明顯減少,而G2期的細胞數則明顯增加。證明以galectin-1蛋白質來處理口腔癌細胞之後,會促使細胞週期加速從S期進行到G2期。
在前面的實驗中發現外加galectin-1蛋白質能促使細胞增生、遷移、和貼附,而anti-Gal-1抗體可抑制口腔癌細胞經由galectin-1所增加的貼附作用,因此接下來以動物模式來測試anti-Gal-1抗體在活體中的治療效果。我們將口腔癌細胞打入免疫缺陷(SCID)的小鼠使其長腫瘤,之後由腹腔注射給與anti-Gal-1抗體觀察腫瘤生長情形,並以免疫組織染色分析血管之分佈情形。結果發現雖然anti-Gal-1抗體對於腫瘤大小並沒有顯著的影響,但是卻能有效減少腫瘤內部的血管。我們也直接以腫瘤注射抗體的方式來治療腫瘤,結果發現直接注射anti-Gal-1抗體確實能夠有效抑制腫瘤的體積。本實驗證明galectin-1蛋白質能夠有效促進口腔癌細胞的遷移、增生、及貼附,並與活化ERK之訊息傳遞有關。進一步以基因晶片及流式細胞儀分析的結果發現,外加galectin-1蛋白質能夠調控與細胞週期有關的基因,並確實對口腔癌之細胞週期造成影響。而動物實驗證明anti-Gal-1抗體可以有效減小腫瘤體積,且能明顯抑制其腫瘤組織中之血管密度。本研究更進一步的釐清外加galectin-1蛋白質對於口腔癌癌化之進程及其分子機制,並指出galectin-1蛋白質或許能成為具有治療腫瘤效果之目標。



英文摘要 Galectins are a family of structurally related carbohydrate-binding proteins, which are defined by their affinity for poly-N-acetyl-lactosamine-enriched glycoconjugates and sequence similarities in the carbohydrate recognition domain. Galectin-1 (Gal-1), a prototype member of this protein family, has been reported that contributes to different events associated with cancer biology, including tumour transformation, cell cycle regulation, cell adhesion, migration and blocking proinflammatory cytokine secretion. In our previous studies, galectin-1 was highly expressed in oral squamous cell carcinoma (OSCC) and especially in their surrounding stroma. In this study, recombinant human galectin-1 was expressed in E. coli system and then purified to investigate its functional characteristics in oral carcinogenesis. Several OSCC cell lines were treated with recombinant galectin-1 proteins and examined their biological activities including proliferation, migration, and adhesion. We found that galectin-1 could enhance oral cancer cells proliferation, adhesion, and migration in a dose dependent manner, and might involve in ERK/Akt pathway. These activities could be blocked with galectin-1 specific antibodies. We also analyzed the cell cycle by flow cytometry and found the cells were increased in G2/M phase when treated with galectin-1. The Oncogene/Kinase microarray profiling was examined to explore the molecular events and to identify candidate genes expressed in OSCC cells responsible for the differential activity mediated by galectin-1. After RT-PCR and Western blotting to further validated expression changes of top 10 upregulated and downregulated genes, we found BRCA1 (breast cancer tumor suppressor gene) and AKIP (Aurora-A kinase interacting protein) were downregulated in galectin-1 treated OSCC cells, and cyclin A1 was upregulated. To further examine the effects of anti-galectin-1 in xenograft tumor model, we found that injected anti-Gal-1 with intraperitoneally did not significantly eradicate the tumor but obviously reduced the vascular densities of tumors. However, the tumor size was significantly reduced when anti-Gal-1 was directly injected the tumor. In conclusion, this study we investigate the possible molecular mechanisms of galectin-1 in oral cancer progression, and it may be as a potential therapeutic target.


論文目次 中文摘要 I
英文摘要 IV
誌謝 VI
目錄 VII
圖目錄 VIII
緒論 1
口腔癌 1
Galectin-1 3
細胞周圍基質對癌細胞所造成的影響 5
細胞週期(cell cycle)與癌症的關係 6
研究動機 7
材料與方法 9
大量表現重組galectin-1蛋白質 9
純化重組galectin-1蛋白質(TALON Resin) 10
純化重組galectin-1蛋白質(FPLC) 11
解凍細胞 12
細胞繼代培養 13
細胞保存 14
細胞計數 15
細胞遷移實驗 Migration Assay 15
Cell Lysates製備 17
SDS-PAGE蛋白質電泳 17
西方點墨法 Western Blotting 19
免疫組織染色 Immunohistochemistry—石蠟包埋組織 21
動物實驗 22
Galectin-1 polyclonal antibody 22
Anti-Gal-1 antibody 腹腔注射對於腫瘤的治療效果 23
Anti-Gal-1 antibody 腫瘤注射對於腫瘤的治療效果 24
細胞增生分析 Cell Proliferation Assay 25
細胞週期染色的分析及量化 26
細胞貼附實驗 Adherence Assay 27
實驗結果 29
討論 37
參考文獻 Reference 44
表 Table 51
圖 Figure 53
附錄一:儀器 73
附錄二:英文縮寫 75
自述 77

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