臺灣博碩士論文加值系統

肺癌是全世界癌症致死的首要原因。其中非小細胞肺癌因個體差異性極大，不論在預後或個人對治療的反應都有顯著差異。因此，若能判別病人是否屬於轉移的高風險族群或能評估其手術後復發率，將對療程有更好的掌控。近年來已有許多研究及越來越多的證據表明，醣基化參與在許多生物反應過程中，當中包含癌症病程轉化和癌細胞轉移等重要機轉。本研究的特定目標是建立一個有效的平台，同時篩選和鑑定肺癌轉移標誌物的血清醣蛋白。首先以刀豆素A親和性層析法純化出肺腺癌患者的血清中的N-醣基化修飾蛋白。得到的醣蛋白經由二維螢光差異電泳法分離再結合奈流液相層析質譜儀分析，最後從數據庫檢索鑑定出蛋白身分。我們鑑定到一些表現量有差異的癌症相關醣蛋白，包括α-1-抗胰蛋白酶，補體C3c，結合球蛋白，上皮鈣粘蛋白等。經由西方墨點法和橙黃網孢盤菌 (Aleuria aurantia)凝集素染色法驗證後，發現包括上皮鈣粘蛋白在內的這些醣蛋白，其核心岩藻醣化的現象隨著肺癌病程進展而增加。我們接著檢測154例肺腺癌病人血清的上皮鈣粘蛋白的岩藻醣化指數（fucosylation index, FI），岩藻醣化指數較低的患者（FI≤ 1.5），其存活率高於岩藻醣化指數較高的患者（FI>1.5）。同時我們建立了一個AlphaLISA檢測平台，量測上皮鈣粘蛋白的岩藻醣化指數。目前的研究結果顯示，血清中上皮鈣粘蛋白的岩藻醣化指數可能是一個有潛力的轉移性肺腺癌的預後指標。除此之外，AlphaLISA檢測平台的發展也已經可以達到許多其他疾病診斷上的高通量篩選需求。

Lung cancer is the leading cause of cancer-associated deaths worldwide. Non-small cell lung cancer is a heterogeneous condition with significant variability in prognosis and in individual response to treatment. Thus, the identification of patients with a high risk of metastasis or relapse after surgery would allow better management. There is increasing evidence that glycosylation plays a significant role in biological processes including oncogenic transformation and metastasis. The specific aim of our study is to set up a platform to screen and identify serum glycoproteins as metastasis biomarkers of lung cancer. Concanavalin A affinity chromatography was used to enrich N-linked glycoproteins from pooled serum of lung adenocarcinoma patients. The captured glycoproteins were separated with 2-D DIGE combined with nano-LC-MS/MS and identified by database searching. Some differentially expressed cancer-related glycoproteins, such as α-1-antitrypsin, complement C3c, haptoglobin, and E-cadherin, were identified. These glycoproteins were evaluated by Western blotting and Aleuria aurantia lectin staining and several, including E-cadherin, showed increased core-fucosylation during lung cancer progression. We then measured the fucosylation index (FI) of E-cadherin in 154 lung adenocarcinoma patients. The survival rate of patient with lower fucosylation index (FI≤1.5) of e-cadherin is higher than that with higher fucosylation index (FI>1.5). In addition, a homogeneous proximity-based AlphaLISA assay to measure the FI of E-cadherin was established. The present study indicates that the FI of E-cadherin could be a potential prognostic marker of metastatic lung adenocarcinoma. Furthermore, the AlphaLISA assay is ready to meet the high throughput screening requirements for the diagnosis of many other diseases.

口試委員會審定書 i
誌謝 ii
中文摘要 iii
Abstract iv
Abbreviation v
Table of Contents viii
ChapterⅠ- Overview and Rationale 1
1.1 Lung cancer: characteristics and diagnosis in the clinic 2
1.2 Lung cancer biomarker discovery using various technologies 3
1.3 Identification of serum glycoprotein biomarkers from human lung adenocarcinoma patients 11
List of Tables 12
Table 1-1. Lung cancer is the leading cause of cancer death in Taiwan 12
Table 1-2. Biomarkers of lung cancer 13
ChapterⅡ-Quantification of serum core-fucosylated E-cadherin as a metastatic lung adenocarcinoma biomarker 14
2.1 Introduction 15
2.2 Experimental Procedures 18
2.3 Results 26
2.4 Discussion 31
Chapter III – Conclusion and perspectives 34
Conclusion and perspectives 35
List of Figures 37
Figure 2-1. AlphaLISA Assay Principle 37
Figure 2-2. Schematic diagram of the glycoproteomic analysis of serum N-glycosylation pattern changes in lung adenocarcinoma. 39
Figure 2-3. Enrichment of glycoproteins by lectin affinity chromatography. 40
Figure 2-4. Differentially expressed glycoproteins. 41
Figure 2-5. Silver-stained 2-DE profiles of the ConA affinity column eluate of pooled serum samples from lung adenocarcinoma patients and normal controls. 42
Figure 2-6. MS/MS spectrum of the corresponding N-glycosylated peptides. 44
Figure 2-7. Screening of fucosylated glycoproteins in the pooled serum samples from normal controls (N) and serum samples from 20 individual patients. 45
Figure 2-8. Survival rates of groups of lung adenocarcinoma patients with a fucosylated index (FI) of E-cadherin > 1.5 or ≧ 1.5 compared by the Kaplan-Meier method. 46

Figure 2-9. The fucosylated index of E-cadherin in patients with early or late stage lung adenocarcinoma was measured by the equation described in experimental procedures. 47
Figure 2-10. AlphaLISA assay. 48
Figure 2-11. Comparison of the FI for E-cadherin in lung cancer sera using the AlphaLISA method or the Western blot and lectin staining method. 50
Figure 2-12. Molecular functions related to non-small cell lung cancer associated with E-cadherin. 51
List of Tables 52
Table 2-1. Clinical features of the patients with lung adenocarcinoma. 52
Table 2-2. Identification of differentially expressed proteins captured by Con A. 53
Table 2-3 N-glycosylation sites of glycoproteins identified by LC-MS/MS 54
Table 2-4. Significance of serum E-cadherin fucosylation in NSCLC patients receiving surgical resection. 55
References 56
Appendix 67
Supplemental Figure S1. The FI index measured ratio on blots is a linear assay. 67
List of instruments 68

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