Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22211
標題: iTRAQ差異蛋白質體分析於癌幹細胞與奈米氧化鋅暴露大鼠之生物標記研究
iTRAQ-Based Comparative Proteomic Analysis of Cancer Stem Cell and Rat Bronchoalveolar Lavage Fluid in Response to ZnO Nanoparticles Exposure
作者: 賴本航
Lai, Ben-Heng
關鍵字: Proteomic;蛋白質體;Cancer Stem Cell;Nanoparticles;iTRAQ;Quantitation;癌幹細胞;奈米氧化鋅;定量
出版社: 分子生物學研究所
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摘要: 
奈米氧化鋅微粒 (nanoparticles zinc oxide) 是目前台灣奈米產業之中積極發展的材料之一,常用於如輪胎、塗料、化妝品、抗菌劑等,可提高耐磨性、延長使用時間、遮蔽紫外線等功能,為具有市場潛力的奈米產品。但奈米氧化鋅進入人體內是否會影響人類健康在目前並不是很清楚,並且與奈米氧化鋅相關的毒理研究並不多,還需更進一步的研究探討。本研究以 Sprague-Dawley 大鼠做為實驗動物,利用微粒產生暴露系統穩定產生粒徑大小約 35 nm 奈米氧化鋅微粒,以總表面積濃度為基準進行呼吸暴露實驗,暴露 6 小時 (n=6) 奈米氧化鋅微粒的為暴露組,並以相同暴露條件下,微粒通過高效能過濾器 (High efficiency particulateair filter,HEPA) 之空氣為對照組 (n=12)。大鼠於暴露後 24 小時進行犧牲並收集肺泡灌洗液 (bronchoalveolar lavage fluid, BALF),再藉由標定 iTRAQ (isobaric tags for relative and absolute quantitation) 同質量試劑的定量蛋白質體學研究方式,以質譜儀 (mass spectrometer, MS) 對暴露組與對照組之蛋白質同時進行定性定量分析,再將經嚴謹定量後的表現量差異蛋白質以 Gene ontology (GO) 進行註解,來探討奈米氧化鋅進入體內的危害機制及差異性蛋白在此機制中扮演的角色,並尋找適合可當生物標記 (biomarker) 的蛋白質,用來做為評估奈米氧化鋅造成傷害的指標。從前人 2D-based 鑑定之 57 個表現量差異蛋白質中,只有 12 個蛋白質與 iTRAQ-based 相同,本研究主要以 iTRAQ-based 的 46 個表現量差異蛋白質進行探討。在發炎及免疫蛋白質當中,S100A8 和 S100A9 可能會同時促使肺部往癌症或肺部纖維化的方向發展,而ITI-H3 則抑制往癌症方面發展,VDBP 則是會激活巨噬細胞進而往慢性阻塞性肺疾病 (COPD) 發展,綜合以上資料,暴露於奈米氧化鋅環境下,可能會促使肺部纖維化進而往慢性阻塞性肺疾病發展。
近幾年的研究發現,在血癌、乳癌和腦癌細胞中存在一群特殊細胞-癌幹細胞,它具有和幹細胞相似的功能並具有侵襲,轉移及多重抗藥性的能力,對於經過手術的癌症病患,體內若殘存著癌幹細胞對於癌症具有極強的復發能力。目前臨床上偵測癌幹細胞的抗體還存在著爭議性,具有偽陽性的疑慮。本研究藉由 MCF-7 乳癌細胞株中的癌幹細胞會表現 ATP-binding cassette transporters (ABCG2) 之特性,可將 Hoechst 33342 染劑快速排出細胞,當進行染色時,癌幹細胞會因此特性而無法染上,經由流式細胞儀分析後會出現 side population,此部分含大多數的癌幹細胞,其他部分則為癌細胞,之後個別萃取細胞內的蛋白質以 iTRAQ 定量蛋白質體技術,找出癌細胞和癌幹細胞之間具有表現差異的蛋白質,再利用 Gene ontology (GO) 的生物資訊方法對具表現量差異的蛋白質進行註解,提供癌幹細胞相關研究、臨床診斷及藥物開發相關資訊。本研究經嚴謹定量分析後鑑定到 76 個表現量差異蛋白質,經 GO 分析後發現,MCF-7 癌幹細胞中分類於糖解作用和檸檬酸循環等能量相關的蛋白質皆為上升表現,而分類在轉譯所需的蛋白質中,大多也是表現量上升的狀況,表示癌幹細胞具有比癌細胞更強的獲得能量和蛋白質合成能力。而這些蛋白質同時具有其他能力可以幫助癌幹細胞,PGI、GAPDH、EF1A2 和 EF-Tu 具有使癌幹細胞有能力躲避凋亡、增加抗藥性、更強的侵襲及轉移能力,另外,粒線體雖然監管了內源性細胞凋亡路徑,但癌幹細胞卻表現許多的 VDAC2 在粒線體外膜上與促凋亡因子結合,使癌幹細胞躲避了內源性的細胞凋亡。
本研究成功的建立並利用 iTRAQ 定量蛋白質體平台對癌幹細胞與肺沖洗液的複雜蛋白質進行研究,提供大量的差異蛋白質資訊,此技術平台也可應用於其他的癌細胞株或是臨床尿液檢體的研究上。
URI: http://hdl.handle.net/11455/22211
其他識別: U0005-1708201122034600
Appears in Collections:分子生物學研究所

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