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標題: | 探討單層奈米碳管與細胞蛋白交互作用及對細胞訊息傳遞之影響 To Identify the Single-walled Carbon Nanotubes Interacted Cellular Proteins and Potential Impacts on Cellular Signaling |
作者: | Chi-Hung Hung 洪啟紘 |
指導教授: | 陳惠文(Huei-Wen Chen) |
關鍵字: | 單層奈米碳管,奈米碳黑,蛋白質暈冕,無同位素標定蛋白質體學分析,改良式BCA蛋白質定量,腺瞟呤核甘酸轉運蛋白,肌動肌凝蛋白,核醣蛋白,核糖核酸解旋?,核糖核酸蛋白粒, single-walled carbon nanotube,nano carbon black,protein corona,labeling free proteomics,modified bicinchoninic acid assay,ADP/ATP translocase,actomyosin,ribosomal protein,RNA helicase,heterogeneous nuclear ribonucleoproteins (hnRNP), |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 獨特的導電性、機械性、熱學性質令奈米碳管變得重要。在半導體、生醫顯像與藥物傳遞上的應用具有極大的潛力的同時,製造奈米碳管的危害和人類暴露到奈米碳管的機會也隨之增加。單層奈米碳管從呼吸道進入肺臟會直接造成細胞毒性和對細胞產生氧化壓力,導致發炎反應。在重複暴露下更會形成肉芽腫而進一步導致纖維化。到目前為止,對於單層奈米碳管在分子層面的潛在毒性和機制仍不清楚。 在本篇研究中,我們藉由蛋白質體學分析蛋白圖譜以探討和單層奈米碳管交互作用的細胞蛋白。在離體實驗中,以肺腺癌上皮細胞A549和人類正常氣管上皮細胞BEAS-2B為蛋白來源分別和單層奈米碳管和奈米碳黑交互作用;蛋白暈冕的強作用力及快速形成顯示碳類奈米物質會和蛋白質形成強蛋白暈冕。在我們的研究中,我們改良BCA (bicinchoninic acid)蛋白質定量試劑來定量樣品緩衝液中的蛋白得以取代胺基酸同位素標定的使用。對單層奈米碳管分別和A549和BEAS-2B細胞蛋白作用的分析結果取交集,總共有503 個蛋白被辨認檢驗。在奈米碳黑的刪減後,剩下179個單層奈米碳管特異性吸附蛋白。蛋白質體結果顯示,單層奈米碳管會增幅濃縮腺瞟呤核甘酸轉運蛋白(ADP/ATP translocase)、肌動肌凝蛋白(actomyosin)和核醣蛋白(ribosomal proteins);在特異性作用的核蛋白中,核糖核酸解旋酶(RNA helicase)和核糖核酸蛋白粒(heterogeneous nuclear ribonucleoproteins)為主要構成。在細胞實驗中,BEAS-2B細胞處理單層奈米碳管後發現誘發細胞型態變異、細胞生長抑制以及細胞週期停滯。綜合以上,我們找到的單層奈米碳管特異性作用蛋白,將為奈米碳管毒性的潛在機制提供新的探討方向。 Carbon nanotubes (CNTs) are very important for their unique electrical, mechanical and thermal properties. With the exceptional potential to the range of semiconductor, biomedical imaging and drug delivery system, the concern of CNTs production and their potential for human to exposure to aerosolized CNTs are rising. Pulmonary toxicity of single-walled carbon nanotubes (SWCNTs) is their direct cytotoxic effect, and induces oxidative stress and inflammatory response upon pharyngeal aspiration or inhalation. After repeatedly exposure of SWCNTs, granuloma will form and lead to fibrosis. Up to now, the potential toxicity and mechanism in molecular biology of SWCNTs are still unclear. We investigated SWCNTs-interacted novel cellular protein by profiling proteins on protein corona and Mass spectrometry-based proteomics. In ex vivo experiments, SWCNTs and nano carbon black (nCB) were interacting with cell lysate (human lung adenocarcinomic alveolar A549 and immortal human bronchial epithelial BEAS-2B cells individually); strong interaction and rapid formation of protein coronas indicated that carbon based nanomaterials interact with proteins as hard corona. In our study, we modified common bicinchoninic acid (BCA) assay to quantify samples in Laemmli buffer in stand of safe isotope labeling amino acid quantification for proteomics. Intersecting of SWCNTs-interacted proteins from A549 and BEAS 2B cell lysate ex vivo, and there are total 503 proteins identified in SWCNT-binding. After eliminated with nCB binding proteins, SWCNTs specific binding list remains 179 proteins. Proteomic results indicated ADP/ATP translocase, actomyosin and ribosomal proteins are amplified by SWCNTs; in the part of nuclear proteins, RNA helicase and heterogeneous nuclear ribonucleoproteins are main member to interact with SWCNTs. In the in vitro part, BEAS-2B cells were induced morphology change, inhibited to proliferate and arrested in cell cycle after SWCNTs treated. In conclusion, we figured out SWCNTs specific interacted proteins, and it will provide a new direction to explore potential mechanisms of toxicity for the carbon nanotubes. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51870 |
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顯示於系所單位: | 毒理學研究所 |
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