利用蛋白質體學技術探討人類口腔癌之唾液生物標幟

Investigation of human salivary biomarkers for oral cancer using proteomics approach

周于楨(Yu-Jen Jou)

指導教授 ： 黃素華 ； 林振文

亞洲大學/健康學院/生物科技學系碩士班/碩士(2009年)

摘要

口腔癌是常見的癌症之一。口腔癌誘因主要為抽菸、喝酒、嚼食檳榔等習慣，與在台灣口腔癌的發生率逐年升高。提早發現口腔癌，使之提高口腔癌的治癒機會。本論文研究目的為將蛋白質體技術分析口腔癌患者唾液中胜肽片段及蛋白，進而得到口腔癌專一性唾液標幟胜肽片段及蛋白。本論文於97年至98年間在中國醫藥大學附設醫院耳鼻喉科採集20個正常者與40個口腔癌患者唾液作為本實驗檢體。首先，我們利用C8-微粒磁珠分離胜肽技術獲得豐富的胜肽片段，再以基質2,5-dihydroxyacetophenone (2,5-DHB)和α-cyano-4-hydroxycinnamicacid ( HCCA ) 將胜肽片段結晶化，然後進行基質輔助雷射脫附游離質譜法 ( MALDI-TOF/TOF MS ) 得到正常及口腔癌患者之胜肽質譜圖。進一步利用ClinProtTM Bruker Daltonic 分析胜肽質譜圖，比較正常組與口腔癌患者之唾液胜肽片差異。在使用2,5-DHB基質結晶化的結果發現4438 Da胜肽為口腔癌患者唾液中常出現胜肽片段，尤其在口腔癌分期為T2患者為41.18％。利用HCCA基質的結晶化結果發現2918 Da胜肽在正常組唾液出現為0 ％，在口腔癌分期T4口腔癌患者唾液為80 ％。利用MALDI-TOF/TOF MS 做單一蛋白質分析 ( MS/MS ) 獲得2918 Da胜肽序列為CNSWEVNLQSISEFIINNRNYSTK，鑑定為zinc finger protein 510 ( ZNF510 )的胜肽片段。以此胜肽序列為抗原製備兔子抗血清，使用於酵素連結免疫吸附分析方法及西方墨點方法確認正常及口腔癌患者唾液2918 Da胜肽的表現量。另一方面，我們也利用二維電泳及基質輔助雷射脫附游離質譜法( MALDI-TOF/TOF MS ) 及單一蛋白質鑑定 ( MS/MS )方法鑑定出transferrin為口腔癌患者唾液表現量較正常組高。經酵素連結免疫吸附分析法和西方墨點法確認transferrin在正常組唾液表現量較低，口腔癌患者唾液表現量較高。本論文鑑定出口腔癌專一性唾液標幟胜肽片段及蛋白，如2918 Da胜肽及transferrin蛋白，可應用於開發口腔癌診斷試劑。

關鍵字

口腔癌 ； 蛋白質體技術 ； 基質輔助雷射脫附游離質譜法

並列摘要

Oral cancer is one of the most frequent cancers worldwide. Tobacco, alcohol and betel quid chewing are the factors for oral cancer, being associated with the increase in the rate of oral cancer incidence in Taiwan. Early diagnosis of oral cancer can provide adequate treatment and increase survival. The goal of the study is to identify oral cancer-specific salivary biomarkers such as peptides and proteins using the proteomic approaches. We collected salivary samples from oral cancer-free individuals and oral cancer patients attended in the China Medical University hospital during 2008 and 2009. Firstly, commercial C8-functionalized magnetic beads were used for salivary peptide separations. C8- affinity peptides were incubated with the matrix 2,5-dihydroxyacetophenone (2,5-DHB) or a-cyano-4-hydroxycinnamic acid (HCCA), and then analyzed using Matrix-Assisted Laser Desoption/Ionization Time-of-flight Mass Spectrometry (MALDI-TOF MS). ClinProtTM Bruker Daltonic analyses revealed that unique mass peak of 4438 Da peptide in 2,5-DHB was in 10% of the saliva from oral cancer-free individuals and 41.18％of the saliva from oral cancer patients with the T2 stage. In addition, unique mass peak of 2918 Da peptide in HCCA was in 0% of the saliva from oral cancer-free individuals and 80％of the saliva from oral cancer patients with the T4 stage. Moreover, MALDI-TOF/TOF MS/MS revealed the amino acid sequence CNSWEVNLQSISEFIINNRNYSTK for 2918 Da peptide, being one fragment of the zinc finger protein 510 (ZNF510). ELISA and Western blotting assays confirmed the amount of 2918 Da peptide in saliva using rabbit polyclonal sera against the 2918 Da peptide. In addition, transferring was identified as the up-regulated protein in the saliva from oral cancer patients using two-dimension gel electrophoresis (2DE) and MALDI-TOF MS. ELISA and Western blotting assays showed a significantly higher protein level in the saliva from oral cancer patients than the saliva from oral cancer-free individuals. The study identified 2918 Da peptide and transferring as oral cancer-specific salivary biomarkers using the proteomic approaches, being helpful for developing the diagnostic kids for oral cancer.

並列關鍵字

oral cancer ； proteomic ； Matrix-Assisted Laser Desoption/Ionization Time-of-flight Mass Spectrometry (MALDI-TOF MS)

參考文獻

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