陳頌方Sung-Fang Chen徐羽薇Yu-Wei Hsu2019-09-042019-7-292019-09-042014http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN060142003S%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/100404肝癌是全球最普遍且致命的癌症之一，而慢性C型肝炎病毒的患者有20~30%會演變為肝硬化甚至是肝癌，由於C型肝炎病毒基因本身變異性大，以致於抗病毒藥物與疫苗的發展都成為醫療界的一大挑戰。本篇研究將藉由HuH7.5-SEAP細胞株模擬C型肝炎病毒顆粒感染細胞，再採用同重元素相對和絕對定量(iTRAQ)的化學標定方法搭配質譜技術，分析在急性與慢性感染情況下其蛋白質相對含量變化。iTRAQ標定的胜肽樣品會以等電聚焦分級分離儀(sIEF)或親水性作用層析法(HILIC)進行分餾，之後進行奈米級液相層析串聯式質譜分析。研究中總共鑑定到2,615個蛋白質，其中1,816個蛋白質具定量結果，並且發現利用二維的液相層析技術來分離胜肽樣品，顯示兩種分離策略能提供良好的互補性與正交性。在急性和慢性感染情況下分別挑選出78和140個具有顯著差異的蛋白質，並以GeneGo生物資訊軟體分析，結果顯示許多蛋白質與細胞骨架重組及細胞吸附等兩種生物作用途徑有相關。目前挑選與胰島素阻抗、囊泡運輸、細胞骨架重組及脂蛋白分泌途徑等相關的蛋白質以西方墨點法(Western blot)進行驗證，期望能發現新穎的蛋白質，並以此作為治療C型肝炎病毒的藥物標記分子。Hepatocellular carcinoma (HCC) is one of the most prevalent and mortal cancer in the world, and 20% to 30% of patients with chronically hepatitis C virus lead to liver cirrhosis and liver cancer. Due to genetic variability of hepatitis C virus, the development of antiviral drugs and vaccines becomes a real challenge. In this study, naïve Huh7.5-SEAP cells were established and infected by hepatitis C virus, then isobaric tags for relative and absolute quantitation (iTRAQ) was applied to investigate protein profiles in both acute and chronic infections. The iTRAQ labeled peptides were fractionated by solution isoelectric focusing (sIEF) or hydrophilic interaction liquid chromatography (HILIC), followed by reversed phase nano-LC tandem mass spectrometry analysis. A total of 2615 proteins were identified, and 1816 of them were also quantified. Two-dimensional liquid chromatography technique that employed on iTRAQ labeled peptides provided results with excellent complementarity and orthogonality. Moreover, 78 and 140 differentially expressed proteins were selected in acute and chronic infection cases for GeneGo analysis. As a result, these proteins were found to be associated with cytoskeleton remodeling and cell adhesion. Besides, proteins correlated with insulin resistance, vesicular traffic, actin remodeling and secretory pathway, will be further validated by Western blot, and could serve as novel diagnostic biomarkers for hepatitis C virus infection.肝癌C型肝炎病毒HuH7.5-SEAP同重元素相對與絕對定量親水性作用層析等電聚焦液相層析串聯式質譜儀hepatocellular carcinoma (HCC)hepatitis C virus (HCV)HuH7.5-SEAPiTRAQHILICisoelectric focusingLC-MS/MS