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系統識別號 U0026-0102201611103600
論文名稱(中文) 非小細胞肺癌與巨噬細胞之介白素6 (IL-6)醣化修飾的差異
論文名稱(英文) Characterization of glycosylation patterns of interleukin-6 between non-small lung cancer cell lines and macrophage
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 104
學期 1
出版年 105
研究生(中文) 黃婷卉
研究生(英文) Ting-Hui Huang
學號 T16021045
學位類別 碩士
語文別 英文
論文頁數 59頁
口試委員 指導教授-蘇五洲
召集委員-張權發
口試委員-林建中
中文關鍵字 肺癌  介白素-6  醣基化 
英文關鍵字 lung cancer  interlukin-6  glycosylation 
學科別分類
中文摘要 根據先前的研究指出,非小細胞肺癌患者在化學治療前,若血液中有高濃度的介白素-6其預後較差,化學治療易使介白素-6濃度更為提高,不利肺癌患者預後情況,因此介白素-6在血液中的濃度對於治療前後的預後評估具有顯著意義。介白素-6主要由T細胞與巨噬細胞所分泌,可視為一種前發炎型細胞激素,能刺激免疫反應引起發炎,甚至進一步造成惡性胸腔積液或促使細胞對化療藥物產生抗藥性。先前研究發現,不同細胞型態所製造的介白素-6,在醣化修飾的位置與程度上具差異性,然而介白素-6的醣化修飾是否影響肺癌細胞的發展依舊是個問號。因此我們去比較肺癌細胞與循環中巨噬細胞所分泌的介白素-6表現量, 我們更嘗試在免疫沈澱實驗中減少抗體輕鏈與介白素-6共存,提昇目標蛋白純度,再以量化巨噬細胞與肺癌細胞培養液中的介白素-6,並利用LC-MS/MS確定蛋白為介白素-6,與Lectin array鑑別各個細胞來源的介白素-6其醣基,藉以尋找出癌細胞的特異性醣基化圖譜。醣化的修飾蛋白在細胞致病過有密切相關,因為醣化修飾在分泌型蛋白為常見的轉錄後修飾,廣泛指在腫瘤相關血清中有醣化的差異,驗明新型的醣化生物標記。生物標記的應用可在於早期危險族群的篩選、早期病理轉換的轉變得整段以及預後治療結果。
英文摘要 We previously reported that NSCLC patients with high circulating interleukin-6 (IL-6) levels had survival outcomes inferior to those of NSCLC patients with low circulating IL-6 levels. Therefore, circulating IL-6 is considered a prognostic factor for patients with NSCLC. In lung cancer patients, the circulating IL-6 can be secreted from tumor cells, inflammatory cells, or others. Because cell-type-specific glycosylation patterns of IL-6 have been detected in previous studies, we wanted to identify the specific glycosylation patterns of IL-6 in lung cancer cells. With these findings, we then will be able to delineate the sources of circulating IL-6 and develop biomarkers for early diagnosis and predictive markers for treatment response in lung cancer patients. In this study, we examined the glycosylation patterns of IL-6 produced from various lung cancer lines, cell lines with specific activated oncogenes, and cells from clinical specimens of lung cancer patients. We then compared IL-6 patterns to those of macrophages. We also amplified lung cancer cell lines and macrophages to collect their conditioned medium, and then tried different experimental conditions to purify 1 g of IL-6 for analysis. We use LC-MS/MS and lectin array to analyze IL-6. Additionally, we planned to develop a method for detecting circulating IL-6 with specific glycosylation patterns. We then hope to be able to evaluate the potential of using lung cancer-specific IL-6 glycoforms as novel biomarkers for early diagnosis and prediction of treatment response in lung cancer patients.
論文目次 中文摘要 I
Abstract II
誌謝 III
Chapter 1 Introduction 1
1-1 The function of Interlukin-6 (IL-6) 1
1-2 The role of glycosylation in cancer 2
1-3 Identification of biomarker for lung cancer 3
1-4 Principle and evolution of immunoprecipitation 4
1-5 Analysis of structural glycomics 5
1-5-1 Glycan identification by Lectin array 6
1-5-2 Protein/glycoprotein identification by LC-MS/MS 6
1-6 Rational 8
1-7 Aim 8
Chapter 2 Materials and Methods 9
2-1 Cell culture 9
2-2 Conditional media (CM) condition 10
2-3 Enzyme-linked immunosorbant assay (ELISA) 11
2-4 Beads conjugation with antibody 11
2-4-1 Dimethyl pimelimidate (DMP) 11
2-4-2 Bis[sulfosuccinimidyl] suberate (BS3) 11
2-5 Immunoprecipitation 12
2-6 Western Blot 13
2-7 Lectin Blot 13
2-8 Gel staining 14
2-8-1 Coomassie brilliant blue stain 14
2-8-2 SYPRO Ruby 14
2-9 LC-MS/MS 14
2-9-1 Immunoprecipitation 14
2-9-2 Trypsin digestion 15
2-9-3 Desalting 15
2-9-4 Mass spectrometry 15
2-10 Lectin array 16
2-10-1 Immunoprecipitation. 16
2-10-2 Extraction 16
2-10-3 Lectin array 16
2-11 Antibodies 17
2-12 Reagents 17
Chapter 3 Results 18
3-1 Expression of IL-6 in macrophage and lung cancer cell lines 18
3-2 Fucosylation in macrophage and lung cancer cell lines 19
3-3 IL-6 purification 20
3-4 Amplification and extraction of IL-6 22
3-5 Identification of IL-6 by LC-MS/MS 23
3-6 Identification of IL-6 glycosyl-pattern by Lectin array 24
Chapter 4 Discussion 26
References 32
Figures 41
Figure 1. Workflow for the glycoproteomic analysis of IL-6 from macrophage and NSCLC cell line 41
Figure 2A. IL-6 production comparison upon various NSCLC cell lines 42
Figure 2B. Production analysis of IL-6 in macrophage from three volunteers 43
Figure 3. IL-6 patterns comparisons between tumor cell lines and macrophages by Western blot and Lectin blot 44
Figure 4. Analysis of IL-6 by (A) Western blot and (B) Coomassie Brilliant Blue stain 45
Figure 5. Cross-linkers for strengthen conjugation antibody on beads 46
Figure 6. Light chain reduction analysis of IL-6 by (A) Western blot and (B) Coomassie Brilliant Blue stain. 47
Figure 7. Efficiency comparison of IL-6 elution with/without 2-ME. 48
Figure 8. IL-6 mass production 49
Figure 9. Determination of (A) IL-6 quantity and (B) IL-6 purity 50
Figure 10. Glycopeptides identification of extracted protein from AS2/I9G by nano LC-MS/MS analysis 51
Figure 11. Glycopeptides identification of extracted protein from macrophage by nano LC-MS/MS analysis 52
Figure 12. Overall glycosylation levels detected by lectin array 53
Tables 54
Table 1 Peptides sequence of the IL-6 from AS2/I9G aligned in the Mascot search engine 54
Table 2 Peptides sequence of the IL-6 from macrophage aligned in the Mascot search engine 55
Table 3. Lectin-Glycan specificity chart 57
Supplementary Information 1 58
Supplementary Information 2 59
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