臺灣博碩士論文加值系統

鼻咽癌是一種主要發生在東南亞地區的癌症，包含新加坡、香港、台灣等等…。由於其耗發在鼻咽的區域，常常造成診斷上的困難，往往診斷出患有鼻咽癌時，患者大多進入了癌症晚期，因而早期診斷早期治療，對於鼻咽癌患者來說是一件相當重要的事情。有鑑於此，我們實驗室兩年前成功利用分泌蛋白質體的技術，期望找出所有可能的鼻咽癌標定蛋白(Wu et al., 2005)，從中挑選了3種可能從鼻咽癌分泌出來的蛋白(BIGH3, CD109 and prosaposin)，來進一步鑑定是否能作為未來鼻咽癌的腫瘤標定蛋白。
鼻咽癌細胞株先於無血清知環境下培養，適當時間後收集上清液，其中所分泌出來的蛋白質，利用8-14%梯度電泳(gradient SDS-PAGE)分離，再進一步利用基質輔助雷射游離時間飛行(matrix-assisted laser desorption/ionization-time of flight, MALDI-TOF) 質譜儀(mass spectrometry)中鑑定身分(Wu et al., 2005)。為了確定其中3種可能的蛋白(BIGH3, CD109 and prosaposin) 是否能作為未來鼻咽癌的腫瘤標定蛋白。首先利用自行合成的3種抗體，成功地偵測到鼻咽癌細胞株所分泌出來的3種蛋白，同時我們也利用免疫組織染色技術(Immunohistochemistry)發現，在大多數鼻咽癌的組織切片檢體中，此3中蛋白在腫瘤組織中皆有大量的表現 (BIGH3: 39/42; CD109: 37/40; prosaposin: 40/41)。為了更進一步達到偵測腫瘤血清標誌的目地，我們亦建立針對BIGH3、CD109 與 prosaposin 的螢光三明治酵素連結免疫分析(fluorimetric sandwich ELISA)平台。利用此平台，我們發現prosaposin在病人血清中的濃度(41.9 ± 16.22 ng/ml, n = 44 )比正常人(21.2 ± 7.63 ng/ml, n = 82)還要來的高(p = 0.014)。
綜合上述分析，prosaposin可能是一個鼻咽癌的腫瘤標誌蛋白。

Nasopharyngeal carcinoma (NPC) is endemic in Southeast Asia including Taiwan, and is commonly diagnosed late because of its deep location and vague symptoms. Therefore, it is important to identify biomarkers for early diagnosis. For this purpose, we have analyzed the secreted proteomes of NPC cell lines (Wu et al., 2005), and 3 of those identified proteins (BIGH3, CD109 and prosaposin) were further examined for their potentials as NPC biomarkers in this study. Secreted proteins of NPC cells were identified by SDS-PAGE combined with MALDI-TOF mass spectrometry. To test whether 3 of the secreted proteins (BIGH3, CD109 and prosaposin) are potential NPC markers, antibodies against the 3 proteins have been produced. All of the 3 proteins were detected in culture media of NPC cells by Western blotting. Then expression of the 3 proteins in tumor tissues was examined by Immunohistochemical analysis; All of the 3 proteins could be found in most of tumor tissues (BIGH3: 39/42; CD109: 37/40; prosaposin: 40/41). The higher level of prosaposin was found in NPC tissues compared to normal tissues (p=0.027). Serum prosaposin level was further detected by sandwich ELISA. Its serum level was higher in NPC (41.9 ± 16.22 ng/ml, n=44 ) than that in healthy control (21.2 ± 7.63 ng/ml, n=82) (p = 0.014). Prosaposin may be a potential biomarker for NPC.

中文摘要 …………………………………………………………………...2
Abstract …………………………………………………………………..3
1. Introduction ……………………………………………….……………4
2. Material and Methods ………………………………………………..8
3. Results
Production of BIGH3, CD109 and prosaposin fusion proteins or peptide polyclonal antibody …………………………………………...18
Detecting BIGH3, CD109, and prosaposin in multiple cell lines secreted proteins………………………………………………………..19
Immunostain of TW02 cell line………………………………………..20
Immunohistochemistry by nude-mouse model, and NPC tissues …..20
Detection of 3 protein by Sandwich ELISA …………………….........21
4. Discussion ………………………………………………………….......23
5. Reference list …………………………………………………………..28
Table ………………………………………………………………………35
Figure legends …………………………………………………………….44
Figure ……………………………………………………………………...51
Appendix ………………………………………………………………….65

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