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研究生:張文馨
研究生(外文):Wen-Hsin Chang
論文名稱:評估蛋白質體學方法用以開發肺腺癌血漿生物標記
論文名稱(外文):Evaluation of several proteomic approaches in discovery of plasma biomarkers for lung adenocarcinoma
指導教授:蔡有光
指導教授(外文):Yeou-Guang Tsay
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:英文
論文頁數:51
中文關鍵詞:肺腺癌血漿生物標記
外文關鍵詞:lung adenocarcinomaplasma biomarkers
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根據世界衛生組織(WHO)統計資料,肺癌是迄今為造成最多癌症死亡的原因。 早期肺癌的五年存活率約為50%,而晚期肺癌則不到5%。肺癌的診斷發現通常已經是晚期階段,也因此預後很差。由於目前尚未有FDA批准的早期肺癌血漿生物標記,因此開發肺癌生物標記仍然是迫切的需求。鑑於我們過去成功的篩選出在某些癌症患者中有表現差異的血漿蛋白質,因此決定使用相同的技術,利用改良式二維電泳檢查肺癌患者的血漿中是否存在任何癌症特異性蛋白質。在第二章中,我們使用巨孔反向層析法,根據蛋白質的疏水性進行第一維分離。接著將第一維分離後不同群的蛋白質進行還原或非還原狀態的聚丙烯酰胺凝膠電泳 (SDS-PAGE)分析。根據這些分析後,發現血清澱粉樣蛋白A-1(SAA1)在肺癌患者的血漿中表現量比正常人高。雖然我們研究血漿蛋白的歷史悠久,但是沒有針對血漿蛋白四級結構的分析,因此,我們嘗試膠體過濾層析法(GFC),分析原始狀態下的血漿蛋白。結果顯示肺腺癌患者的血漿樣品中存在具有~100 千道爾吞的SAA1四級結構。我們還發現在卵巢癌或系統性紅斑狼瘡患者中有些微的SAA1訊號,這與之前文獻中指出除了肺癌外只有少部分疾病有表現SAA1訊號的結果一致。有趣的是,我們發現肺癌患者中有幾個額外的SAA1信號。簡而言之,我們將繼續探討SAA1蛋白的差異性表現是否具有作為肺癌特異性蛋白質生物標記的潛力。此外,由於微陣列研究顯示在肺組織中α2-巨球蛋白(α2M)有高表現量,我們對血漿中α2M的四級結構特別感興趣。根據目前的文獻,α2M應該是由四個180 千道爾吞次單元組成720千道爾吞的血漿蛋白。我們的研究結果也顯示α2M四級結構可能包含四個180 kDa次單元,並且這些次單元是以分子間雙硫鍵鍵結。總而言之,我們可以利用這些篩選平台開發其他疾病的蛋白質生物標記。
Currently, according to World Health Organization, lung cancer (LC) is the number one cancer killer. Five-year survival is about 50% for early stage LC, but only < 5% in advanced one. Most patients are diagnosed with advanced LC, and the prognosis of LC is poor (Woodard et al., 2016). Since there are still no FDA-approved early plasma biomarker for LC, development of biomarkers for LC still has a pressing need. Given our past success in identification of plasma protein species from patients with certain cancers, we decided to use the same technique, modified 2-D DIGE, to examine whether any cancer-specific proteins were present in the plasma from patients with lung adenocarcinoma (LUAD). In chapter II, we used macroporous reverse phase (mRP) chromatography for first dimensional separation of proteins according to their hydrophobicity. Proteins in the mRP fractions were analyzed using reducing or non-reducing SDS polyacrylamide gel electrophoreses for second dimensional separation. We found that serum amyloid A-1 (SAA1) protein expressed higher in the plasma samples of LUAD patients according to the proteomic analyses based on mRP chromatography. Although we have been studying plasma proteins for a long time, there was no systemic characterization of quaternary structures of these plasma proteins. Thus, we employed gel filtration chromatography to resolve plasma proteins of interest at their native states. Through this strategy, we have discovered that there is an SAA1 quaternary structure with molecular mass of ~100 kDa in the plasma specimen from LUAD patients. In addition, SAA1 in the plasma of lung cancer patients is not eluted together with high-density lipoprotein complexes. We also found that much lower SAA1 expression in plasma from patients with ovarian cancer or systemic lupus erythematosus, consistent with the observation that a few diseases have low SAA1 expression. Interestingly, we found that there were several extra SAA1 signal in LUAD patients. In short, we will verify whether SAA1 has the potential to serve as LC-specific protein biomarker. Moreover, we are particularly interested in quaternary structure of α2-macroglobulin (α2M protein) in plasma since microarray analyses showed its high expression in lung tissue. According to current literature, α2M is known to be a 720-kDa plasma protein that is supposed to be a tetrameric complex. Our data are consistent with the model that the α2M quaternary structure may probably contain four of ~180-kDa subunits, and these subunits are connected using intramolecular disulfides. We also found several additional α2M quaternary structures are present in the plasma from lung cancer patients as well as control individuals. More importantly, with these screening platforms for cancer biomarker, we are able to identify more protein quaternary structures in various diseases.
TABLE OF CONTENTS
誌謝......I
中文摘要.......III
ABSTRACT.........IV
TABLE OF CONTENTS........VI
LIST OF TABLES........VII
LIST OF FIGURES.........VIII
LIST OF ABBREVIATIONS....X
CHAPTER I LITERATURE REVIEW.....1
1.1 INTRODUCTION FOR LUNG CANCER.........2
1.1.1 Epidemiology......2
1.1.2 Screening and diagnosis......2
1.1.3 Clinical treatment.......11
1.1.4 Early detection for lung cancer........12
1.2 METHODOLOGY OF PROTEOMIC ANALYSIS OF LUNG CANCER.......13
CHAPTER II Identification and verification of SAA1 protein as a potential lung cancer biomarker using various chromatographic techniques.....16
SUMMARY......17
INTRODUCTION.....18
MATERIALS AND METHODS.......20
RESULTS.....24
DISCUSSION.....38
APPENDIX........41
REFERENCES......47
SUPPLEMENT.......50

LIST OF TABLES
CHAPTER Ⅰ
Table 1. Classification of lung cancer.......7
Table 2. Staging for small cell lung cancser.......8
Table 3. TNM staging system for non-small cell lung cancer.......9
Table 4. Staging system for non-small cell lung cancer.......11


LIST OF FIGURES
CHAPTER Ⅱ
Figure 1. The workflow for analysis of differential signal from plasma of patients with lung adenocarcinoma through modified two-dimensional difference gel electrophoreses.......29
Figure 2. Fluorescence analyses of the plasma samples from patients and healthy individuals following modified two-dimensional difference gel electrophoreses.......30
Figure 3. LC-MS/MS analyses of the differential signal in figure 2.......31
Figure 4. Western blot analysis of serum amyloid A-1 (SAA1) in plasma samples.......32
Figure 5. The gel filtration chromatography separation of plasma samples.......33
Figure 6. Western blot analysis of serum amyloid A-1 (SAA1) in indicated GF fractions.......34
Figure 7. Western blot analysis of apolipoprotein A-1 (ApoA1) in indicated GF fractions.......35
Figure 8. The gel filtration chromatography (GFC) separation of plasma samples.......36
Figure 9. Western blot analysis of serum amyloid A-1 (SAA1) in indicated GF fractions.......37
APPENDIX
Figure 10. Gene expression of α-2-macroglobulin by various tissues in human.......41
Figure 11. Surface model of humanα-2-macroglobulin.......42
Figure 12. Anti α2-macroglobulin western blot analysis of plasma proteins present in indicated GF fractions.......43
SUPPLEMENT
Supplement Figure 1. Fluorescence analyses of the plasma samples from patients and healthy individuals following modified two-dimensional difference gel electrophoreses.......49
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