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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48761

Title: 癌細胞株於低氧環境所誘導之變異選擇性剪接蛋白質體學研究
Hypoxia-Induced Alternative Splicing Variants Proteomics in Cancer Cells
Authors: Chia-Hsun Liu
劉家薰
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 癌細胞缺氧;選擇性剪接;蛋白質體學;液相層析串聯式質譜儀;胜肽序列資料庫
Alternative splicing;Hypoxia;Cancer cell;Mass spectrometry-based proteomics;Peptide library
Date: 2016
Issue Date: 2018-08-22T06:31:04Z
Abstract: 腫瘤細胞急速分裂生長時,其心細胞與組織常面臨缺乏血液及養份供給的狀態,使周圍形成相對低氧之微環境 (tumor hypoxia),當腫瘤半徑增殖大於200 μm時細胞便會開始進行細胞凋亡路徑,因此通常存在許多因應機制來適應此缺氧環境,其中低氧誘發轉錄蛋白 (HIF) 已被許多研究證實為缺氧環境下最主要之基因調控者,特別會誘導基因產生非規律性之選擇性剪接作用影響生理機制,但過去變異選擇性剪接研究皆針對轉錄體學層次分析,無法直接證實變異轉錄體實際產生各種蛋白質異構體於細胞內執行功能,而近幾年以高解析度質譜儀為基礎之蛋白質體學研究快速發展,大幅增進鑑定蛋白質之準確度與靈敏度,然而質譜數據分析需依賴資料庫進行比對,而現今標準蛋白資料庫大多提供生物體於正常生理情況下分析,缺乏許多變異異構體 (isoform) 資訊,因此本研究透過Ensembl上取得之基因序列資訊以及生物資訊處理技術,建構一分析特定選擇性剪接之胜肽序列資料庫,特別專注於外顯子跳躍與內隱子保留剪接模式,稱為Novel_KR,並將其應用於鑑定人類乳癌細胞株 (MCF7) 於低氧逆境環境下產生之選擇性剪接變異蛋白質異構體的身分,於此分別將樣品搭配上兩種不同類型之蛋白質體學前處理模式,為膠體輔助蛋白質水解法搭配一維高效液相層析分離系統與SDS-PAGE一維分離搭配上膠內蛋白質水解法,隨後即以Waters™品牌之SYNAPT G2 MS與Thermo Scientific™品牌之LTQ Orbitrap液相層析串聯式質譜儀 (LC-MS/MS) 進行分析,所得之初始數據經轉檔後,以Mascot演算軟體進行理論與實際之二次圖譜的比對,並將輸出之結果執行圖譜編碼過濾與重複次數篩選,即可得出鑑定之變異胜肽所對應的基因名稱清單,藉由Innate DB與DAVID軟體進行生理功能與參與路徑搜尋,接著將結果清單資訊透過與過去研究文獻搜尋比對以及設計所屬之引子 (primer) 執行即時定量聚合酶鏈鎖反應,提供選擇性剪接變異之蛋白質異構體的存在證據,且驗證於轉錄體層次之轉譯發生。本研究從比對結果鑑定出缺氧情況下四重複以上之獨特胜肽片段共計11條,將個別所對應之基因經查詢後,其中有三個基因過去與缺氧調控相關,分別為TTN、ATXN1、LRP1基因,其分別產生之變異異構體參與心臟內之功能、VEGF基因之活化、細胞存活之傳導路徑的調控相關,間接增加了癌細胞之轉移與侵略能力,然而以定量即時聚合酶鏈鎖反應執行變異選擇性剪接轉錄體之驗證時,其結果無法與蛋白質體相呼應,但後續會持續針對多種獨特變異基因設計引子,進行轉錄體試驗,並期望能搭配上同位素標定執行剪接變異蛋白質異構體之表現量差異分析,希冀能釐清癌細胞面臨缺氧壓力時,如何改變生理作用與調節選擇性剪接機制,使基因與蛋白質發生生成變動,最後即可透過整合人類乳癌細胞株之全蛋白質體輪廓,提供腫瘤生物學相關研究有利的新資訊。
Alternative splicing, a tightly regulated process during gene expression in eukaryotes, produces mature RNA variants coding for multiple proteins from a single gene. This pivotal mechanism can significantly increase the biodiversity of proteins from the same genome. When organisms encounter stresses, the alternative splicing mechanism is triggered and results in the production of various spliced protein isoforms. According to recent studies in cancer biology and developmental biology, hypoxia-inducible factor (HIF) induced or non-HIF induced genes were regulated through alternative splicing when cells are under hypoxia. However, the current studies of alternative splicing were mainly derived from the perspective of genomics or transcriptomics, but the point of view from comprehensive proteomics were lack because of no available database for MS data analysis. In this study, we construct an alternative splicing database and develop a mass spectrometry-based method for qualitatively profiling proteins from variant alternative splicing. A novel protein database which contains two types of alternative spliced peptide will be constructed through bioinformatics and be evaluated by mass spectrometry-based proteomic techniques. The specific aims is to develop a method for identifying proteins from alternative splicing based on mass spectra data in particular in exon skipping and intron retention. Once the analytical method and database is established, the aim plans to globally characterize proteins from differential splicing events when human breast cancer cell line MCF7 is under hypoxia. We also perform transcriptomic study to further confirm the existence of proteins with alternative spliced isoforms. Therefore, by means of our newly developed method, the hypoxia-induced alternative spliced proteome can lead us to further understand how organisms response to hypoxia. We believe that our novel method will provide a new direction for alternative splicing-omics studies in the future.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010336006.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48761
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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