鼻咽癌為中國人特有之疾病，由於鼻咽癌發生位置於鼻腔深處，往往發現時已進入中末期。因此，要找到可診斷出早期鼻咽癌的生物指標的確有其存在的必要性。分泌蛋白對於癌症的形成扮演著一重要角色，又利用分泌蛋白可進入人體血液循環之特性，可做為未來癌症篩檢與偵測上之應用，但目前在分泌蛋白樣本收集上仍缺乏一有效方法。因此本研究目的便是以蛋白質體學方法來分析以中空纖維管柱生物反應器系統所收集而來之鼻咽癌細胞分泌蛋白，並從中尋找可能生物指標。將細胞以無血清培養液培養於中空纖維管柱生物反應器系統之中，再進一步以此系統對細胞之分泌蛋白做收集。為確保以此系統所收集到之樣本實為細胞所分泌，利用西方墨點法比較等細胞數下，細胞所分泌出之蛋白以及胞內蛋白中肌動蛋白含量之差異，並利用此結果推估出細胞死亡數低於0.037%以下。接著以SDS-PAGE及液相層析電噴灑串聯式質譜儀對分泌蛋白樣本加以鑑定，結果找到134個蛋白樣本；首先藉由蛋白是否重複出現於細胞內篩選出108個蛋白，接下來再以signal peptide、transmembrane domain的有無篩選出82個蛋白，之後以生物資訊方法對各個蛋白之功能作更進一步篩選，選擇和鼻咽癌特性、癌症功能相關之蛋白，最後篩選出integrin beta-4、CUB and sushi domain-containing protein 2、N-acetylglucosamine-6-sulfatase、legumain、laminin subunit alpha-5、glypican-1、beta-mannosidase、agrin、lysosomal protective protein、thrombospondin-1十個分泌蛋白作為可能之鼻咽癌生物指標候選者。利用中空纖維管柱生物反應器系統可有效對細胞之分泌蛋白做收集，再結合蛋白質體學分析技術運用在尋找癌症生物指標上，未來更可針對這十個鼻咽癌生物指標候選者做更進一步之驗證。

Nasopharyngeal carcinoma (NPC) is endemic in southern China. Diagnosis of NPC is intricate because its morbid region is located in deep nasal cavity, resulting in the diagnosis of later-stage NPC. Therefore, biomarkers of NPC are of instant demands for the detection of NPC in an early stage. Secretome, secreted from cells into the blood circulation, plays a critical role in cancer progression and can be applied to biological monitoring or screening of cancers. But now, it lacks effective methods to collect secreted proteins. The objective of this study is to use the proteomic technique to analyze NPC cell secretome collected from a hollow fiber bioreactor system for biomarker discovery. To avoid serum contamination, NPC cells were adapted into serum-free medium. The hollow fiber bioreactor system where cells were cultured was utilized to collect secretome of NPC cell line. In order to make sure that collected proteins were mainly from secretion rather than cell lysis, actin levels of intracellular and extracellular samples were evaluated by Western blot to estimate cell death number. At best, only 0.037% of cells may lyse. For identification of secretome, the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) technology and nano-HPLC-ESI-MS/MS were used. Total 134 proteins were identified in NPC cell secretome, of which, 108 proteins were not observed in cell lysate. Among those 108 proteins, about 76% are known to be secreted due to containing signal peptide(s) and/or transmembrane domain(s). Ten proteins, integrin beta-4, CUB and sushi domain-containing protein 2, N-acetylglucosamine-6-sulfatase, legumain, laminin subunit alpha-5, glypican-1, beta-mannosidase, agrin, lysosomal protective protein, and thrombospondin-1 were considered as biomarker candidates according to their protein functions, such as peculiar NPC-related ones, cancer-related adhesion, metastasis, and so on. This approach may be useful for collection of secretome and discovery of novel biomarker candidates for clinical application.

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