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系統識別號 U0026-2108201305140300
論文名稱(中文) 藉由分析血清中氮基醣鏈找尋口腔癌之癌症標記
論文名稱(英文) Serum N-glycans profiling for the discovery of potential oral cancer tumor markers
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 101
學期 2
出版年 102
研究生(中文) 古世昀
研究生(英文) Shih-Yun Guu
學號 T36004089
學位類別 碩士
語文別 英文
論文頁數 63頁
口試委員 口試委員-謝達斌
口試委員-陳玉玲
口試委員-陳怡婷
指導教授-張權發
中文關鍵字 口腔癌  氮基醣鏈  生物標記 
英文關鍵字 N-glycan  biomarker  oral cancer 
學科別分類
中文摘要 醣化是一種重要的蛋白質轉譯後修飾,蛋白質的醣化對其功能有很大的影響。研究指出蛋白質上醣類結構的改變和癌症的發生及轉移有密切的關係,而這些醣類結構的改變也可以作為檢測癌症的生物標記。在此篇研究中,我們藉由分析正常人和口腔癌病患血清中的氮基醣鏈找尋口腔癌之癌症標記。血清中的蛋白質經過冷凍乾燥、化學還原(reduction)、烷化(alkylation)後,再經由胰蛋白酶(trypsin)處理,得到胜肽(peptide)片段。接著利用氮基醣水解酶F (peptide N-glycosidase F, PNGase F),從胜肽片段中獲得氮基醣鏈,接著利用碳18固相萃取匣(C18 Sep-Pak cartridge)進行純化。最後將純化的氮基醣鏈進行全甲基化保護(permethylation),再利用基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF-MS)以正離子(positive ion)模式進行分析,得到血清中氮基醣鏈的質譜。我們藉由軟體分析質譜,並預測出相對應於質譜上可能的醣類結構,接著利用統計方法,分析血清中以結構區分的氮基醣鏈群以及特定結構氮基醣鏈的相對表現量。結果發現,在口腔癌病患的血清中,經過岩藻醣化(fucosylation)和唾液酸化(sialylation)修飾的三分支及四分支結構的氮基醣鏈有較高的相對表現量。另外,我們也篩選出了在口腔癌病患血清中出現相對表現量變化,並具有75%以上診斷準確度(accuracy)的7種氮基醣鏈。這些在口腔癌病患血清中所發現有顯著相對表現量變化的氮基醣鏈,相信可以做為未來發展口腔癌之生物標記的參考指標。
英文摘要 Glycosylation is a protein post translational modification which plays important role in protein function, stabilization, as well as turnover. Alteration of protein glycosylation is reported to be associated with tumorigenesis and metastasis and is demonstrated to be potential cancer biomarkers. In this study, serum N-glycomes from normal populations and oral cancer patients were analyzed for the discovery of potential oral cancer biomarker. Total serum proteins were lyophilized and subjected to chemical reduction, alkylation and trypsin digestion. The N-glycans were released by treated glyco-peptides with PNGase F and purified by Sep-Pak C18 cartridge. After permethylation, the purified N-glycans were analyzed using MALDI-TOF-Mass spectrometry in positive ion mode to generate a serum N-glycan profile. The possible N-glycan structures were predicted from Mass spectrum by GlycoWorkbench and the relative abundance of these N-glycans were calculated statistically. We found that some tri-antennary and tetra-antennary glycans with varying degrees of fucosylation and sialylation was increased in serum N-glycome of oral cancer. We also found that the relative abundance of 7 N-glycans decreased or increased in the serum sample of oral cancer with diagnostic accuracy greater than 75%. Hence, these serum N-glycans identified from oral cancer patients should be considered as the candidates of potential oral cancer biomarkers.
論文目次 Contents
中文摘要 I
Abstract II
Acknowledgement III
Contents IV
List of Figures VI
List of Tables VIII
Abbreviations IX
Chapter 1 Introduction 1
1-1 Oral Cancer 1
1-2 Glycosylation 1
1-3 Altered glycosylation pattern in cancer 3
1-4 Glycomics 4
Chapter 2 Study objective and specific aim 7
Chapter 3 Experimental Procedures 8
3-1 Serum Samples 8
3-2 N-glycan release and purification 8
3-3 Permethylation of N-glycans 9
3-4 MALDI-TOF MS analysis 9
3-5 Data evaluation 10
Chapter 4 Results 11
4-1 MS analysis of N-glycans released from the serum glycoproteins 11
4-2 Serum N-glycan profiling for seeking of oral cancer biomarkers 11
4-2.1 Analysis of specific glycan structures 12
4-2.2 Analysis of different glycan subclasses 15
4-2.3 Correlation between serum N-glycans with the progression of oral cancer 16
4-2.4 Correlation between serum N-glycans and lymphatic metastasis of oral cancer 16
Chapter 5 Discussions 18
5-1 Alteration of serum N-glycans in oral cancer 18
5-2 The serum samples in this thesis 19
5-3 The altered glycosylation pattern in oral cancer cell line 20
5-4 MS/MS analysis of identified glycans 20
Conclusion 21
References 22
Figures 28
Tables 53
Appendix-1 59
Appendix-2 60
Appendix-3 61
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