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研究生:張玉薇
研究生(外文):Yu-Wei Chang
論文名稱:以蛋白質體整合性分析細胞和核內與肺癌細胞轉移相關之蛋白質
論文名稱(外文):Identification of metastasis-related proteins by integrative analysis of lung cancer cell lysate and nuclear proteomes
指導教授:吳韋訥
指導教授(外文):Wailap Victor Ng
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暨研究所
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:2010
畢業學年度:98
語文別:中文
論文頁數:56
中文關鍵詞:肺癌轉移蛋白質體
外文關鍵詞:lung cancermetastasisproteomics
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肺癌為全世界常見造成高致死率的癌症之一,肺癌主要可分成兩大類,一類為非小細胞肺癌 (大約佔所有肺癌種類的百分之八十五) ,另一類為小細胞肺癌 (大約佔所有肺癌種類的百分之十五)。肺癌的五年存活率僅僅只有百分之十五,原因在於當病人被診斷為肺癌時,癌細胞通常已經轉移出去。因此,找尋參與在非小細胞肺癌轉移有關的蛋白質顯得格外重要;我們利用四株非小細胞肺癌的細胞株 (非轉移性的細胞株-CL1-0與H23,轉移性細胞株-CL1-5和H838) 為研究模型來探討參與在肺癌轉移相關的蛋白質為何。我們分別萃取這四株細胞株的全細胞溶解與核蛋白質體,將其利用串連式質譜儀作分析;藉由分析量化這兩組具有不同轉移能力細胞株的核蛋白與蛋白質體,我們可以找到一組在轉移細胞株特異性表達且可能參與轉移相關的候選蛋白質。目前為止,大約有243個SILAC標記的CL1-0和CL1-5全細胞溶解蛋白質、453個CL1-0全細胞溶解蛋白質、476個CL1-5全細胞溶解蛋白質、491個H23全細胞溶解蛋白質與446個H838全細胞溶解蛋白質被質譜儀偵測。利用非標記的量化方式,我們總共找到了123個在CL1-5轉移性細胞中特異性表達的蛋白質。為了得到作為後續診斷試劑開發的理想候選參與肺癌轉移的蛋白質,我們將所有質譜儀偵測到的特異性表達的蛋白質與已公開的轉錄體資料相互比對。因此,我們篩選出17個參與肺癌轉移相關的候選蛋白質。在這17個候選蛋白質中,我們找到了一個新穎的蛋白質為人類OLA1,此蛋白質在過去曾被報導與乳癌轉移有關 (Jiawei Zhang et al., 2009)。藉由過去文獻上的記載,或許我們可以進一步去探討人類OLA1在非小細胞肺癌的轉移上扮演甚麼角色。我們由衷希望我們初步的研究分析對於臨床的癌症診斷會有幫助。
Lung cancer is one of the most common fatal cancers worldwide. The two major types of lung cancer are non–small cell lung cancer (NSCLC; about 85% of all lung cancer cases) and small-cell lung cancer (about 15%). The 5-year survival rate for all stages combined in NSCLC is only 15% because it often spreads to other parts of the body before it is found. Therefore, the discovery of metastasis-related candidates in NSCLC is of vital importance. In this study, we used four lung cancer cell lines including the non-metastatic CL1-0 and H23, and the highly metastatic CL1-5 and H838 as models to discover the invasion-associated proteins via comparative analysis of their cell lysate, nuclear, and cytosolic proteomes by tandem mass spectrometry and then examining their expression profiles using publicly available microarray data. Approximately 243 SILAC labeled CL1-0 and CL1-5 (12,138 peptides) , 453 unlabeled CL1-0 (9,241 peptides), unlabeled 476 CL1-5 (10,779 peptides), 491 unlabeled H23 (16,937 peptides) and 446 unlabeled H838 (14,272 peptides) human cell lysate proteins have been identified by MS/MS. Approximately 123 proteins were identified as up-regulated protein candidates in CL1-5 cell line by label-free quantitation. To obtain a list of metastasis-related candidates for further development of diagnostic reagents, those differential expressed proteins in highly metastatic cell lines were examined against publicly available microarray datasets. Upon an integrative analysis of 920 lung cancer up-regulated genes from publicly available microarray datasets and the 123 up-regulated proteins in CL1-5 cell line, 17 metastasis-associated candidates were identified. Among the metastasis-associated candidates, we found a protein, hOLA1 (human Obg-like ATPase 1, also call GBP45) which was found to be associated with breast cancer cell migration in the previous study. The presence of invasiveness associated in my candidate list suggest the proteins identified in my study may worth further examination of their role in cancer metastasis and the possibility of developing diagnostic reagents.




Contents
Abstract (Chinese)………1
Abstract…………………………………………2-3
1.Introduction………………………4-9
1.1 Lung cancer…………………………………4
1.2 Metastasis in lung cancer…………………………5
1.3 Metastasis-related marker discovery by proteomics approach……5-7
1.4 Integrative analysis of proteomic and microarray data………7-8
1.5 Gene functions and tumorigenesis mechanisms of hOLA1………………8
1.6 Specific aim………………………………………………9

2. Materials……………………………………..10-12
3. Methods…………………………………13-17
4. Results……………………………………………18-23
4.1 Analyses of the NSCLC nuclear and cytoplasmic proteomes by LC-MS/MS...18-19
4.2 Label-free (peptide counting) and SILAC-based quantitative analyses of non-metastatic and metastatic lung cancer cell proteomes……..……………19-20
4.3 Protein interaction network analysis………………………………………………21-22
4.4 Integrative proteomics and microarray analysis of lung cancer cell proteomes and microarray data……………………….22
4.5 hOLA1 may be a metastasis-related marker in lung cancer cells……………22-23
5. Discussion…………………………24-25
6. References…………………………………………26-29
7. Figures…………………30-39
8. Tables-------------------------------.40-45
9. Appendices……………………………………….46-56

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