跳到主要內容

臺灣博碩士論文加值系統

(3.95.131.146) 您好!臺灣時間:2021/07/28 23:52
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:林睿昌
研究生(外文):Ruey-Chang Lin
論文名稱:氧化鋅奈米微粒及其複合物之細胞毒性研究
論文名稱(外文):Cytotoxicity study of zinc oxide hybrid nanoparticles
指導教授:鍾仁傑鍾仁傑引用關係
口試委員:曾靖孋林忻怡蔡偉博
口試日期:2012-01-17
學位類別:碩士
校院名稱:國立臺北科技大學
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:2012
畢業學年度:100
語文別:中文
論文頁數:80
中文關鍵詞:氧化鋅奈米微粒細胞毒性體外細胞染色細胞周期
外文關鍵詞:Zinc oxide composite nanoparticleCytotoxicityIn vitroCell stainingCell cycle
相關次數:
  • 被引用被引用:0
  • 點閱點閱:171
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
目前奈米材料相關產品在市場上愈趨廣泛與精巧,奈米材料類似於次細胞結構增加與細胞反應性,甚至對環境及生物產生潛在的威脅,因此有必要深入探討奈米微粒與生物系統之交互作用。本實驗以氧化鋅奈米微粒 (ZnO)、氧化鋅結合二氧化矽 (ZnO/SiO2)、氧化鋅結合聚乙二醇 (ZnO/PEG)、氧化鋅結合二氧化矽並結合聚乙二醇 (ZnO/SiO2-PEG) 為材料,以 L929 細胞株 (L929 cell)進行實驗。H&E 與 DAPI 染色觀察細胞形態及細胞核變化;CCk-8 及 LDH Kit分析細胞毒性;流式細胞儀分析細胞週期、凋亡、壞死與攝取奈米微粒含量百分比;膠體電泳 (SDS PAGE) 檢視蛋白質變化。本研究所獲致結果如下:
一、 ZnO 於20 μg/ml劑量時產生許多空泡,40 μg/ml 細胞大量死亡呈現濃度依勢性質,ZnO-PEG,濃度大於 150 μg/ml 才具明顯毒性。
二、 ZnO/SiO2-PEG 奈米複合物,經由細胞主動攝取奈米微粒 (endocytosis) 於高濃度短時間與低濃度長時間,明顯呈現奈米微粒於細胞內部化 (internalization) 現象且具濃度與時間之依恃性質。
三、 細胞週期反應出奈米微粒使 DNA 受到傷害,S 期停滯現象,細胞需要多時間來修復受損之 DNA 以防止細胞產生突變。
四、 高濃度奈米微粒造成細胞凋亡與壞死變化,經由 Annexin V 與PI 螢光染色呈現在流式細胞儀偵測資料上。
五、 細胞回應奈米微粒之蛋白質變化,呈現在蛋白質電泳 (SDS PAGE) 圖上。
依據研究結果,可提供生醫檢測參考,奈米藥物設計新策略與治療系統應用資訊。


In the present days, nanotech-based consume products are widespread and sophisticated. However, the size of nanoparticle is similar to the sub-cell structures so that it may lead to severe reactions with the cells which are potentially hazard to the environment and the organisms. It is urgent and necessary to deeply investigate the interactions between nanoparticle and cell. In this study, Zinc oxide nanoparticle (ZnO), zinc oxide/silicon dioxide composite nanoparticle (ZnO/SiO2), zinc oxide nanoparticle coated with polyethylene glycols (ZnO/PEG) and zinc oxide/silicon dioxide composite nanoparticle coated with polyethylene glycols (ZnO/SiO2-PEG) were used to study their interactions with biological system. L929 cell-line was cultured for the in vitro study. H&E staining and DAPI staining assays were carried out to observe the morphological changes of whole cell and nuclear; CCk-8 and LDH Kit analyses were used to study the cytotoxicity; quantification of cell cycle, apoptosis, necrosis, and cell uptake of nanoparticles were analyzed through the flow cytometer; and gel electrophoresis (SDS PAGE) was done to view the protein changes. The results including:
a. Under a 20 μg/ml dose of ZnO, the cells presented a number of vacuoles. Above 40 μg/ml, cell viability decreased dramatically in a dose-dependent manner. For ZnO-PEG, a concentration greater than 150 μg/ml leaded to apparent toxicity.
b. For ZnO/SiO2-PEG, the cells presented active endocytosis and the internalization of the nanoparticles were observed in a dose-dependent manner.
c. The cell cycle analyses showed that the nanoparticles leaded to damages of DNA, arrest of S phase and more time for restoring the damages.
d. High concentration of nanoparticle leaded to changes in the ratio of apoptosis and necrosis. Annexin V and PI fluorescent staining showed the differences through the flow cytometry analyses.
e. Through protein electrophoresis (SDS PAGE), the changes of protein in the cells responding to the nanoparticles were presented.


中文摘要.................................................................................................................i
英文摘要................................................................................................................ii
誌謝.......................................................................................................................iii
表目錄 ....viii
圖目錄 .....ix
第一章 緒論........................................................................................................1
1.1 奈米科技的演進.....................................................................................1
1.2 研究背景與動機.....................................................................................2
第二章 文獻探討................................................................................................4
2.1奈米之意涵及奈米材料定義..................................................................4
2.2奈米科技的應用......................................................................................5
2.3奈米材料之毒性對細胞影響..................................................................7
2.4奈米微粒與人體健康關係......................................................................7
2.5細胞生長與死亡....................................................................................10
2.6氧化鋅奈米微粒及其複合物之簡介....................................................17
第三章 材料與方法..........................................................................................18
3.1 實驗儀器及藥品...................................................................................18
3.2實驗方法及流程....................................................................................20
3.2.1 氧化鋅奈米微粒及其複合物之製備........................................20
3.2.2 細胞培養....................................................................................21
3.2.3 细胞染色及細胞形態觀察........................................................23
3.2.4 細胞毒性分析............................................................................26
3.2.5 流式細胞儀分析........................................................................29
3.2.6 蛋白質含量測定及凝膠電泳分析............................................34
3.2.7 統計分析方法............................................................................39
第四章 結果與討論..........................................................................................40
4.1 細胞形態觀察分析...............................................................................40
4.1.1 Hematoxylin-Eosin Stain............................................................40
4.1.2 DAPI Nucleic Acid Stain............................................................44
4.2 細胞活性分析.......................................................................................47
4.2.1 CCK-8 cell counting Kit 分析...................................................47
4.2.2 LDH Cytotoxicity Detection Kit 分析.......................................50
4.3 流式細胞儀分析...................................................................................52
4.3.1細胞攝取奈米微粒之螢光強度分析.........................................52
4.3.2 細胞攝取奈米微粒濃度與時間之螢光強度分析....................58
4.3.3 細胞周期分析 (Cell cycle analysis).........................................61
4.3.4 Annexin V-FITC 和 PI 雙染色................................................64
4.4 蛋白質電泳...........................................................................................72
結論......................................................................................................................75
參考文獻..............................................................................................................76


[1]馬振基(2003)。奈米材料科技原理及應用:全華圖書公司。
[2]S. Al-Bader, J. Craig, A. Dunn et al., "Nanoscience and nanotechnologies: opportunities and uncertainties," T. R. S. T. R. A. o. Engineering, ed., The Royal Society & The Royal Academy of Engineering, 2004.
[3]S.-C. LEE, and S.-M. TANG, “The Regulation of the Potential Risks to Nanotechnology in Taiwan,” S. C. Lee, S.M. Tang / Asian Journal of Management and Humanity Sciences, vol. 1, no. 2, pp. 293-308, 2006.
[4]阮國棟、汪芷嫣、林琮禧、吳婉怡、郭家成(2005)。分子奈米科技的環境效益,第二屆環境保護與奈米科技學術研討會
[5]李翰杰(2006)。奈米微粒風險評估之探討:國立中山大學環境工程研究所。
[6]鄭尊仁、林宜平、劉蓉燕、李梅菁、簡弘民(2006)。奈米微粒健康風險評估與管理研究:以奈米氧化鋅為例 研究成果報告 (完整版):行政院國家科學委員會專題研究計畫。
[7]賴炤銘、李錫隆(2003)。奈米材料的特殊效應與應用。CHEMISTRY(THE CHINESE CHEM. SOC., TAIPEI),61(4),585-597。
[8]陳浩銘、劉如熹、胡淑芬(2005)。金屬奈米粒子特性及其製作技術介紹。
[9]張立德(2003)。奈米材料的主要應用領域。檢自:http://tainano.com/chin/nanomaterial%20applications%201.htm
[10]H.-E. Wichmann, C. Spix, T. Tuch et al., Daily Mortality and Fine and Ultrafine Particles in Erfurt, Germany Part I: Role of Particle Number and Particle Mass, WA 754 R432, Health Effects Institute, 2000.
[11]W.-X. Zhang, “Nanoscale iron particles for environmental remediation: An overview,” Journal of Nanoparticle Research, vol. 5, pp. 323-332, 2003.
[12]D. M. Brown, V. Stone, P. Findlay et al., “Increased in flammation and intra- cellular calcium caused by ultra carbon black is independent of transition metals or other soluble components,” Occup Environ Med vol. 57, no. 10, pp. 685-691, 2000.
[13]M. D. Butterworth, L. Illum, and S. S. Davis, “Preparation of ultrafine silica- and PEG-coated magnetite particles,” Colloids and Surfaces A, vol. 179, pp. 93-102, 2001.
[14]張振平、陳春萬 (2009)。不同型態奈米微粒之製作與細胞株或動物暴露系統開發與測試研究:行政院勞工委員會勞工安全衛生研究所。

[15]Y. Zhang, L. Hu, D. Yu et al., “Influence of silica particle internalization on adhesion and migration of human dermal fibroblasts,” Biomaterials, vol. 31, no. 32, pp. 8465-74, Nov, 2010.
[16]W. Song, J. Zhang, J. Guo et al., “Role of the dissolved zinc ion and reactive oxygen species in cytotoxicity of ZnO nanoparticles,” Toxicol Lett, vol. 199, no. 3, pp. 389-97, Dec 15, 2010.
[17]L. Sun, Y. Li, X. Liu et al., “Cytotoxicity and mitochondrial damage caused by silica nanoparticles,” Toxicol In Vitro, vol. 25, no. 8, pp. 1619-29, Dec, 2011.
[18]楊秀宜(2005)。金屬燻煙熱(metal fume fever):勞工安全衛生研究所。
[19]M. Geiser, S. Schurch, and P. Gehr, “Influence of surface chemistry and topo- graphy of particles on their immersion into the lung''s surface-lining layer,” J Appl Physiol, vol. 94, no. 5, pp. 1793-801, May, 2003.
[20]G. Oberdorster, A. Maynard, K. Donaldson et al., “Principles for chara- cterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy,” Part Fibre Toxicol, vol. 2, pp. 8, Oct 6, 2005.
[21]S. van den Heuvel, “Cell-cycle regulation,” WormBook, pp. 1-16, 2005.
[22]J. Groenendyk, and M. Michalak, “Endoplasmic reticulum quality control and apoptosis,” Acta Biochimica polonica, vol. 52, no. 2, pp. 381-395, 2005.
[23]H.-J. Rode, D. Eisel, and I. Frost, Apoptosis, Cell Death, and Cell Pro- liferation, Roche Applied Science.
[24]A. Elsaesser, and C. V. Howard, “Toxicology of nanoparticles,” Adv Drug Deliv Rev, Sep 8, 2011.
[25]G. Faria, C. R. Cardoso, R. E. Larson et al., “Chlorhexidine-induced apoptosis or necrosis in L929 fibroblasts: A role for endoplasmic reticulum stress,” Toxicol Appl Pharmacol, vol. 234, no. 2, pp. 256-65, Jan 15, 2009.
[26]S. Gupta, A. Agrawal, S. Agrawal et al., “A paradox of immunodeficiency and inflammation in human aging: lessons learned from apoptosis,” Immun Ageing, vol. 3, pp. 5, 2006.
[27]邱鼎鈞(2010)。氧化鋅奈米結構合成及特性分析。臺北市:國立臺北科技大學。
[28]Z. Wang, X. Kong, Y. Ding et al., “Semiconducting and Piezoelectric Oxide Nanostructures Induced by Polar Surfaces,” Advanced Functional Materials, vol. 14, no. 10, pp. 943-956, 2004.
[29]T. H. Chung, S. H. Wu, M. Yao et al., “The effect of surface charge on the uptake and biological function of mesoporous silica nanoparticles in 3T3-L1 cells and human mesenchymal stem cells,” Biomaterials, vol. 28, no. 19, pp. 2959-66, Jul, 2007.
[30]B. C. Heng, X. Zhao, S. Xiong et al., “Toxicity of zinc oxide (ZnO) nanoparticles on human bronchial epithelial cells (BEAS-2B) is accentuated by oxidative stress,” Food Chem Toxicol, vol. 48, no. 6, pp. 1762-6, Jun, 2010.
[31]S.-H. Wu, Y.-S. Lin, Y. Hung et al., “Multifunctional mesoporous silica nano- particles as dual-mode imaging probes,” in From Zeolites to Porous MOF Materials – the 40th Anniversary of International Zeolite Conference, 2007, pp. 1804.
[32]H. Yang, C. Liu, D. Yang et al., “Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: the role of particle size, shape and composition,” J Appl Toxicol, vol. 29, no. 1, pp. 69-78, Jan, 2009.
[33]W. Lin, Y. W. Huang, X. D. Zhou et al., “In vitro toxicity of silica nano- particles in human lung cancer cells,” Toxicol Appl Pharmacol, vol. 217, no. 3, pp. 252-9, Dec 15, 2006.
[34]A. K. Jayaraman, and S. Jayaraman, “Increased level of exogenous zinc induces cytotoxicity and up-regulates the expression of the ZnT-1 zinc trans- porter gene in pancreatic cancer cells,” J Nutr Biochem, vol. 22, no. 1, pp. 79-88, Jan, 2011.
[35]Y. Ye, J. Liu, J. Xu et al., “Nano-SiO2 induces apoptosis via activation of p53 and Bax mediated by oxidative stress in human hepatic cell line,” Toxicol In Vitro, vol. 24, no. 3, pp. 751-8, Apr, 2010.
[36]S. Panigrahi, and D. Basak, “ZnO–SiO2 core–shell nanorod composite: Microstructure, emission and photoconductivity properties,” Chemical Physics Letters, vol. 511, no. 1-3, pp. 91-96, 2011.
[37]H.-M. Xiong, Y. Xu, Q.-G. Ren et al., “Stable Aqueous ZnO@Polymer Core- Shell Nanoparticles with Tunable Photoluminescence and Their Application in Cell Imaging,” J. AM. CHEM. SOC, vol. 130, no. 24, pp. 7522-7523, 2008.
[38]F. M. Veronese, and G. Pasut, “PEGylation, successful approach to drug delivery,” Drug Discovery Today, vol. 10, no. 21, pp. 1451-1458, 2005.
[39]C. Yague, M. Moros, V. Grazu et al., “Synthesis and stealthing study of bare and PEGylated silica micro- and nanoparticles as potential drug-delivery vectors,” Chemical Engineering Journal, vol. 137, no. 1, pp. 45-53, 2008.
[40]G. Nabiyouni, A. Barati, and M. Saadat, “Surface Adsorption of Polyethylene Glycol and Polyvinyl Alcohol with Variable Molecular Weights on Zinc Oxide Nanoparticles,” Iranian Journal of Chemical Engineering, vol. 8, no. 1, 2011.
[41]Q. He, J. Zhang, J. Shi et al., “The effect of PEGylation of mesoporous silica nanoparticles on nonspecific binding of serum proteins and cellular responses,” Biomaterials, vol. 31, no. 6, pp. 1085-92, Feb, 2010.
[42]S. Liufu, H. Xiao, and Y. Li, “Investigation of PEG adsorption on the surface of zinc oxide nanoparticles,” Powder Technology, vol. 145, no. 1, pp. 20-24, 2004.
[43]H. Xu, F. Yan, E. E. Monson et al., “Room-temperature preparation and characterization of poly(ethylene glycol)-coated silica anoparticles for biomedical applications,” Journal of Biomedical Materials Research vol. 66A, no. 4, pp. 870-879, 2003.
[44]R. A. Bozym, F. Chimienti, L. J. Giblin et al., “Free zinc ions outside a narrow concentration range are toxic to a variety of cells in vitro,” Exp Biol Med (Maywood), vol. 235, no. 6, pp. 741-50, Jun, 2010.
[45]J. Blummel, N. Perschmann, D. Aydin et al., “Protein repellent properties of covalently attached PEG coatings on nanostructured SiO(2)-based interfaces,” Biomaterials, vol. 28, no. 32, pp. 4739-47, Nov, 2007.
[46]Y. Li, L. Sun, M. Jin et al., “Size-dependent cytotoxicity of amorphous silica nanoparticles in human hepatoma HepG2 cells,” Toxicol In Vitro, vol. 25, no. 7, pp. 1343-52, Oct, 2011.
[47]A. Neumeyer, M. Bukowski, M. Veith et al., “Propidium iodide labeling of nanoparticles as a novel tool for the quantification of cellular binding and uptake,” Nanomedicine, vol. 7, no. 4, pp. 410-9, Aug, 2011.
[48]M. Y. Shie, S. J. Ding, and H. C. Chang, “The role of silicon in osteoblast-like cell proliferation and apoptosis,” Acta Biomater, vol. 7, no. 6, pp. 2604-14, Jun, 2011.
[49]M. Xu, D. Fujita, S. Kajiwara et al., “Contribution of physicochemical characteristics of nano-oxides to cytotoxicity,” Biomaterials, vol. 31, no. 31, pp. 8022-31, Nov, 2010.
[50]M. R. Lorenz, V. Holzapfel, A. Musyanovych et al., “Uptake of functionalized, fluorescent-labeled polymeric particles in different cell lines and stem cells,” Biomaterials, vol. 27, no. 14, pp. 2820-8, May, 2006.
[51]M. S. Cartiera, K. M. Johnson, V. Rajendran et al., “The uptake and intra- cellular fate of PLGA nanoparticles in epithelial cells,” Biomaterials, vol. 30, no. 14, pp. 2790-8, May, 2009.
[52]Y. Hu, J. Xie, Y. W. Tong et al., “Effect of PEG conformation and particle size on the cellular uptake efficiency of nanoparticles with the HepG2 cells,” J Control Release, vol. 118, no. 1, pp. 7-17, Mar 12, 2007.
[53]B. De Berardis, G. Civitelli, M. Condello et al., “Exposure to ZnO nano- particles induces oxidative stress and cytotoxicity in human colon carcinoma cells,” Toxicol Appl Pharmacol, Apr 29, 2010.

[54]C. He, Y. Hu, L. Yin et al., “Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles,” Biomaterials, vol. 31, no. 13, pp. 3657-66, May, 2010.
[55]C. C. Huang, R. S. Aronstam, D. R. Chen et al., “Oxidative stress, calcium homeostasis, and altered gene expression in human lung epithelial cells exposed to ZnO nanoparticles,” Toxicol In Vitro, vol. 24, no. 1, pp. 45-55, Feb, 2010.
[56]J. Chantal , and M. H. Ratinaud, “Cell cycle analysis by flow cytometry: Principles and applications,” Biol Cell vol. 78, pp. 15-25, 1993.
[57]曾厚、張佑民、何佳安、黃世宏、李明威、陳志宏 等(2007)。生醫奈米技術。在教育部生醫奈米科技教學資源中心編著。
[58]B. I. Tarnowski, F. G. Spinale, and J. H. Nicholson, “DAPI AS A USEFUL STAIN FOR NUCLEAR QUANTITATION,” Biotechnic & Histochemistry, vol. 66, no. 6, pp. 296-302, 1991.


連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top