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研究生:楊昆翰
研究生(外文):Kun-HanYang
論文名稱:透過IL-6刺激點帶石斑魚之蛋白質體分析發現DNM2在魚類免疫上扮演之角色
論文名稱(外文):Through the proteomics analysis in IL-6 stimulated orange-spotted grouper (Epinephelus coioides) to discover DNM2 takes important role in fish immunity
指導教授:林翰佑
指導教授(外文):Han-you Lin
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:156
中文關鍵詞:點帶石斑魚免疫系統細胞激素介白素-6蛋白質體學IPA生物資訊學
外文關鍵詞:Epinephelus coioidesIL-6proteomicsbioinformaticsIPA
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有關IL-6的功能仍然在探索階段,在過去的研究揭露IL-6會誘發各種非特異性免疫反應,並在肝臟產生例如補體、非特異性抗體、急性反應蛋白抑或抗菌蛋白等抗菌物質,以增進對於入侵病原的抵抗力。然而對於其中機轉知知甚少。為了瞭解有那些免疫路徑參與其中,本研究令蛋白體學為可能的免疫因子分析。利用串聯質譜儀進行非標定散彈槍式蛋白質體分析,發現IL-6刺激的魚隻共有五百種蛋白質的表現量俱顯著上升。再運用生物資訊預測生物反應路徑,發現與病毒感染時所誘發的相關途徑具高度相關。我們再次以qRT-PCR進一步分析mRNA表現量,得知DNM2的表現不論在轉錄階段或是轉譯階段皆具顯著差異。同時利用不同的免疫刺激後,DNM2 的表現也與蛋白質體所鑑定之趨勢相符。並且在比對不同的臟器表現,發先在肝臟的DNM2的反應在表現反應量或是持續時間比其他免疫器官更為明顯。因此,我們認為在免疫刺激下DNM2與IL-6有一定關聯,並且這個反應可能為肝臟特有的反應。至於哪些分子的表現可能與DNM2的表現及功能相關,則是未來可以進一步研討的亮點。
The reseach of the function of IL-6 in Epineohelus coioides is still at the exploratory stage, our previous studies revealed that IL-6 can induce non-specific immune responses. In grouper’s liver IL-6 will stimulate the production of complement, non-specific antibody, acute phase protein and AMPs, which enhance the antipathogen ability, but little we known about the mechanism in this phenomenon. To further investigate the immunal factors involved in it, we use tandem mass spectrometry to analyze IL-6 stimulated liver by label-free shotgun proteomics. We identified 414 proteins out of 1100 are significantly regulated. We further use IPA bioinformatics tool to process our proteomics dataset. Finding it is highly correlated to viral infection pathway. qRT-CPR is used to further investigate the mRNA expression level, DNM2 has significant difference under both transcriptional and translational level. Also, the mRNA fold change is correlated to our proteomics dataset under different immunal stimulants. Furthermore, comparing DNM2 expression in immunal organs, liver has higher expression level and longer expression period than others. According to this study, we confirm expression of DNM2 under immunal stimulation correlates to IL-6; this phenomenon may be liver-specific. However, the molecules involved in DNM2 expression and its role in IL-6 stimulation needs further research.
中文摘要............................................. I
英文摘要............................................ II
誌謝............................................... VI
目錄.............................................. VII
表目錄.............................................. X
圖目錄.............................................. XI
縮寫表.............................................XIII
一、研究背景.......................................... 1
1-1點帶石斑魚......................................... 1
1-2介白素-6 (interleukin-6; IL-6)................... 2
1-3 IL-6在點帶石斑魚的重要性........................... 4
1-4 免疫相關反應刺激物................................. 7
1-5 蛋白質體學 (Proteomics) 的發展與重要性................9
1-6 生物資訊分析.......................................11
1-7 Dynamin的相關研究.................................12
1-8 研究目的...........................................14
二、材料與方法..........................................15
2-1 點帶石斑魚檢體收集...................................15
2-2 臟器免疫相關聯之基因表現量初篩.........................17
2-3 蛋白質粗萃取與濃度測定................................20
2-4 蛋白質上機前處理.....................................22
2-5 二維電泳分析........................................23
2-6 nLC-Q-Orbit MS/MS質譜儀分析及資料庫比對 .............25
2-7 蛋白質體資料校正....................................26
2-8 Ingenuity Pathway Analysis分析...................26
三、結果...............................................27
3-1 rIL-6刺激後點帶石斑魚之免疫相關臟器基因表現量...........27
3-2 二維電泳分析........................................28
3-3 nLC-Q-Orbit MS/MS上機資料分析......................29
3-4 IPA分析..........................................30
3-5 real-time PCR驗證蛋白質體之表.......................31
3-6 脂多醣體LPS刺激後對DNM2 mRNA表現量之影響...............32
3-7 刀豆凝集素ConA刺激後對DNM2 mRNA表現量之影響.............32
3-8 植物血球凝集素PHA刺激後對DNM2 mRNA表現量之影響...........33
四、討論................................................34
4-1 蛋白質體及生物資訊對本研究之影響........................34
4-2 rIL-6刺激後點帶石斑魚肝臟蛋白質體可能影響之基因挑選.......35
4-3 模擬細菌感染對DNM2之影響..............................36
4-4 模擬淋巴細胞聚集對DNM2之影響...........................37
4-5 研究結論............................................38
參考文獻................................................40
圖表...................................................52
附錄...................................................75

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