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系統識別號 U0026-0812200911413570
論文名稱(中文) 以M13 噬菌體呈現技術來鑑定與 galectin-1 結合之胜肽
論文名稱(英文) Identification of galectin-1 binding peptides by M13 phage display
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 93
學期 2
出版年 94
研究生(中文) 蘇信忠
研究生(英文) Shin-Jung Su
電子信箱 puyo.puyo@msa.hinet.net
學號 t4691104
學位類別 碩士
語文別 中文
論文頁數 105頁
口試委員 指導教授-陳玉玲
口試委員-謝達斌
口試委員-洪澤民
召集委員-靳應台
中文關鍵字 None 
英文關鍵字 oral cancer  galectin-1  phage display  RON 
學科別分類
中文摘要   口腔癌是指包含嘴唇、舌頭、臉頰、嘴巴及口咽部其他部位的癌症總稱,是一種與抽煙、喝酒和嚼食檳榔有關的侵犯性癌症,病患通常在晚期才會表現出徵狀,且在治療之後,復發的機率很高。蛋白質體學是近年來發展的分析技術,對尋找癌化相關的腫瘤標記以作為人類疾病的診斷、治療與預防等研究助益很大。Q. Y. He等人以蛋白質體分析口腔舌癌相關蛋白的表現,發現galectin-1在舌癌的表現與周圍其他正常黏膜組織有明顯的差異。但目前對於galectin-1在口腔內的致癌機制仍不清楚。
  本實驗乃利用噬菌體呈現技術來鑑定與galectin-1結合之胜肽,期能對galectin-1的致癌機制的解析有所助益。經過四次的噬菌體呈現技術篩選,我們從一隨機表現12個胜肽序列的噬菌體庫中,找到許多能辨識galectin-1蛋白的噬菌體。我們比較這些胜肽的親和性,DNA序列分析結果,發現其中兩段序列在篩選中重覆出現,分析比對人類的基因庫之後,發現Met oncogene相關的tyrosine kinase, RON(recepteur d'origine nantais)具有此兩段序列,我們進而利用免疫共沉澱的方式評量galectin-1與RON(及其相關蛋白Met)間之交互作用,不過無法看到galectin-1與RON或Met免疫共沉澱的現象。然而,以共軛焦顯微鏡觀察galectin-1與RON在HSC-3細胞中表現的情形,可以發現galectin-1與RON具有co-localization的現象,且RON免疫組織染色的結果,亦可看到其在口腔腫瘤組織中的表現情形與galectin-1相似。此外,我們也以哺乳動物雙雜交(Mammalian two- hybrid system)來進一步證明噬菌體呈現所篩選出來之胜肽與galectin-1間的交互作用,結果顯示luciferase活性並沒有明顯的改變。總結來說,利用噬菌體呈現技術我們找到一些galectin-1結合胜肽並分析其氨基酸序列及結合強度,其中兩個出現多次的galectin-1結合胜肽的氨基酸序列與RON的氨基酸序列具高度相似性,雖然我們無法以免疫共沉澱法證明galectin-1與RON間的直接交互作用,但是從免疫染色中,包括腫瘤細胞及組織確實有許多徵兆顯示RON與galectin-1間有相關性,galectin-1在口腔癌癌化的作用機制是否透過RON或其他分子的交互作用還需要進一步實驗加以評估,這些galectin-1結合胜肽或許可作為口腔癌研究的有效工具及當作診斷或治療口腔癌策略之參考。
英文摘要  Oral cancer is defined to include cancers of the lip, tongue, cheek, other sites of the month, and the oropharynx. Oral carcinoma is an aggressive tumor that particularly affects chronic smokers, drinkers and betel quid chewers. Patients often present symptoms at a late stage, and there is a high recurrence rate after treatment. Proteomics is a powerful analytical technology newly developed to identify the molecular makers that are associated with malignant transformation, and also to enhance our study of the diagnosis, treatment and prevention of human diseases. Q. Y. He et al. have reported the first proteomic analysis of oral tongue carcinoma to globally search for tumor related proteins. A number of tumor-associated proteins including galectin-1 were consistently found to be significantly altered in their expression levels in tongue carcinoma tissues, compared with their paired normal mucosa. But the mechanism of galectin-1 protein on the oral carcinogenesis is still poorly understood.
 The aim of this study is to identify the binding peptides of galectin-1 using phage display technology. After four rounds of panning, several phage plaques that recognized galectin-1 protein were obtained from the 12-mer phage-displayed peptide libraries. The binding affinities of these selected phages to galectin-1 have been compared. DNA sequencing indicated that two peptides sequence displayed on some of the selected phages with high frequency. BLAST analysis against human gene database showed that these two displayed peptide sequences have homology to the sequence of RON, a c-met-related tyrosine kinase protein. The interaction of galectin-1 and RON (and also its related protein, Met) was evaluated by co-immunoprecipitation, but galectin-1 couldn’t co-immunoprecipitate with these two proteins in our results. Co-localization of RON proteins and galectin-1 proteins had been observed in HSC-3 cells by confocal immunofluorescence staining. We also observed that the expression pattern of RON in oral cancer was similar to that of galectin-1 by immunohistochemistry. Mammalian two-hybrid system was used to further demonstrate the interaction between galectin-1 and the selected peptides in cell culture system. Our data showed that there were no significant changed in the luciferase activity of cells transfected with pCMX-VP16-galectin-1. In summary, several galectin-1 binding peptides have been obtained by phage display screening and characterized their binding affinity. Two frequently present peptides bear the sequences with highly homology to that of RON. Although we failed to demonstrate the direct binding of RON and galectin-1 by co-immunoprecipitation, but there were many indications that showed the interaction between RON and galectin-1. The roles of the galectin-1 and RON in oral carcinogenesis need more experiments to explore and understand. The Galectin-1-binding peptides might be a powerful tool for the study of oral carcinogenesis and might open way for discovery of small molecules with diagnostic and therapeutic value on oral squamous carcinoma.
論文目次 誌謝....1
中文摘要....3
英文摘要....5
目錄....7
圖目錄....9
表目錄....10
緒論....11
1.口腔癌....11
1.1前言....11
1.2口腔癌的發生率....11
1.3口腔癌簡介....12
1.4口腔癌的臨床分期....13
1.5口腔癌預防篩檢之重要性....15
1.6口腔癌的研究與蛋白質體學....15
2.Galectin-1....17
2.1Galectin-1簡介....17
2.2Galectin-1與癌症的關係....18
3.Phage display....21
3.1噬菌體呈現技術簡介....21
3.2噬菌體呈現胜肽庫(peptide library)....22
3.3噬菌體呈現的流程....22
3.4噬菌體呈現技術的應用....23
4.研究動機....24
材料與方法....25
生物親和性篩選(Bio-panning)....25
噬菌體增殖(Amplify)....27
噬菌體效價之測定(Titer)....28
單一噬菌體之挑選與保存....29
噬菌體單股DNA之萃取....31
測定binding activity....32
解凍細胞....33
細胞繼代培養....34
細胞保存....35
細胞計數....36
Cell lysate製備....36
免疫沉澱法....38
SDS-PAGE蛋白質電泳....39
西方墨點法(Westen blotting)....41
細胞螢光染色....42
免疫組織染色....44
構築pCMX-VP16-galectin-1....45
構築pCMX-Gal4-Ph.D.7與pCMX-Gal4-Ph.D.10....47
哺乳動物雙雜交(Mammalian two-hybrid)....49
結果....51
討論....57
參考文獻....66
圖....79
表....97
附錄....101
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