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研究生: 李哲宇
Che-Yu Li
論文名稱: 以螢光奈米鑽石探討斑馬魚胚胎的細胞質動態
Fluorescent Nanodiamond as a Probe for Exploring Cytoplasmic Dynamics in Zebrafish Embryo
指導教授: 張煥正
Chang, Huan-Cheng
林震煌
Lin, Cheng-Huang
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2012
畢業學年度: 100
語文別: 中文
論文頁數: 49
中文關鍵詞: 螢光奈米鑽石斑馬魚胚胎卵黃細胞質流顯微注射即時單一粒子追蹤
英文關鍵詞: fluorescent nanodiamonds, zebrafish embryo, cytoplasmic streaming of yolk, microinjection, real-time single particle tracking
論文種類: 學術論文
相關次數: 點閱:292下載:8
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  • 近來在生物顯影應用方面,螢光奈米鑽石已發展成一項新穎的工具。由於此材料具有許多獨特性質,包括高生物相容性、易於將生物分子修飾在表面的特性以及良好的光穩定性,使得鑽石作為活體外和活體內的光學奈米探針十分具有前景。此篇論文以斑馬魚為生物模型,利用此新穎奈米材料的光穩定且無毒性的特性,作為在生物活體內追蹤工具。我們以顯微注射裝置將螢光奈米鑽石注入單細胞時期斑馬魚胚胎的卵黃細胞,進行長時間的追蹤及觀察細胞質流的運動過程,接著利用單一粒子追蹤技術來探查粒子的運動。我們在卵黃細胞內細胞質錯綜複雜的運動中,觀測到單一方向性且時行時止的運輸方式。並且在早期的斑馬魚胚胎發展階段中(發育1–2個小時的受精卵),測定40個粒子於胚胎中隨著軸向的細胞質流移動的速率,其範圍在0.19 – 0.40 μm/s之間。

    Fluorescent nanodiamonds (FNDs) have recently developed into an exciting new tool for bioimaging applications. The material possesses several unique features including high biocompatibility, easy bioconjugation, and perfect photostability, making it a promising optical nanoprobe both in vitro and in vivo. This work explores the potential application of the novel nanomaterial as a photostable, nontoxic tracer in vivo using zebrafish as a model organism. We introduced FNDs into the yolk cell of a zebrafish embryo by microinjection at the 1-cell stage. Movements of the injected particles were investigated by using single particle tracking techniques. We observed unidirectional and stop-and-go traffic as part of the intricate cytoplasmic movements in the yolk cell. We determined a velocity in the range of 0.19 – 0.40 μm/s for 40 particles moving along with the axial streaming in the early developmental stage (1 – 2 hpf) of the zebrafish embryos.

    Abstract II 摘要 III 目錄 IV 表目錄 VI 圖目錄 VI 第一章 緒論 1 1.1 前言 1 1.2 螢光奈米鑽石 2 1.2.1 鑽石的結構特性 2 1.2.2 鑽石的缺陷中心 5 1.3 斑馬魚 7 1.3.1 以斑馬魚作為脊椎動物模式生物 8 1.3.2 斑馬魚的胚胎發育 12 1.3.3 斑馬魚早期胚胎的卵胞質分離 18 1.4 研究動機 20 第二章 實驗方法 22 2.1 藥品 22 2.2 儀器 23 2.3 螢光奈米鑽石製備 27 2.4 螢光奈米鑽石修飾牛血清白蛋白 29 2.5 螢光光譜測量 30 2.6 斑馬魚培養及胚胎收集 30 2.7 長時間影像觀測的斑馬魚胚胎固定 31 2.8 顯微注射 32 2.9 胚胎影像 33 第三章 結果與討論 34 3.1 螢光奈米鑽石 34 3.2 螢光奈米鑽石於斑馬魚之生物分佈 37 3.3 螢光奈米鑽石於斑馬魚胚胎之細胞質動態 42 第四章 結論 44 第五章 參考文獻 45

    1 Johnson, I. Review: Fluorescent probes for living cells. The Histochemical Journal 30, 123-140, doi:10.1023/a:1003287101868 (1998).
    2 Tsien, R. Y. The green fluorescent protein. Annu Rev Biochem 67, 509-544, doi:10.1146/annurev.biochem.67.1.509 (1998).
    3 Lippincott-Schwartz, J., Altan-Bonnet, N. & Patterson, G. H. Review: Photobleaching and photoactivation: following protein dynamics in living cells. Nature Cell Biology 5, S7–S14, doi:10.1038/ncb1032 (2003).
    4 Michalet, X. et al. Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics. Science 307, 538-544, doi:10.1126/science.1104274 (2005).
    5 Chan, W. C. W. & Nie, S. Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection. Science 281, 2016-2018, doi:10.1126/science.281.5385.2016 (1998).
    6 Pinaud, F., Clarke, S., Sittner, A. & Dahan, M. Probing cellular events, one quantum dot at a time. Nat Meth 7, 275-285 (2010).
    7 Derfus, A. M., Chan, W. C. W. & Bhatia, S. N. Probing the Cytotoxicity of Semiconductor Quantum Dots. Nano Letters 4, 11-18, doi:10.1021/nl0347334 (2003).
    8 Nirmal, M. et al. Fluorescence intermittency in single cadmium selenide nanocrystals. Nature 383, 802-804 (1996).
    9 Mahler, B. et al. Towards non-blinking colloidal quantum dots. Nat Mater 7, 659-664 (2008).
    10 Schrand, A. M. et al. Are Diamond Nanoparticles Cytotoxic? The Journal of Physical Chemistry B 111, 2-7, doi:10.1021/jp066387v (2006).
    11 Nguyen, T. T.-B., Chang, H.-C. & Wu, V. W.-K. Adsorption and hydrolytic activity of lysozyme on diamond nanocrystallites. Diamond and Related Materials 16, 872-876, doi:10.1016/j.diamond.2007.01.030 (2007).
    12 Yu, S.-J., Kang, M.-W., Chang, H.-C., Chen, K.-M. & Yu, Y.-C. Bright Fluorescent Nanodiamonds:  No Photobleaching and Low Cytotoxicity. Journal of the American Chemical Society 127, 17604-17605, doi:10.1021/ja0567081 (2005).
    13 Fu, C.-C. et al. Characterization and application of single fluorescent nanodiamonds as cellular biomarkers. Proceedings of the National Academy of Sciences 104, 727-732, doi:10.1073/pnas.0605409104 (2007).
    14 Davies, G. & INSPEC. Properties and growth of diamond. (INSPEC, the Institution of Electrical Engineers, 1994).
    15 Davies, G. & Hamer, M. F. Optical Studies of the 1.945 eV Vibronic Band in Diamond. Proceedings of the Royal Society of London. Series A, Mathematical and Physical Sciences 348, 285-298 (1976).
    16 Lawson, S. C., Fisher, D., Hunt, D. C. & Newton, M. E. On the existence of positively charged single-substitutional nitrogen in diamond. Journal of Physics: Condensed Matter 10, 6171-6180, doi:10.1088/0953-8984/10/27/016 (1998).
    17 Rand, S. C. & DeShazer, L. G. Visible color-center laser in diamond. Opt. Lett. 10, 481-483 (1985).
    18 Mita, Y., Nisida, Y., Suito, K., Onodera, A. & Yazu, S. Photochromism of H2 and H3 centres in synthetic type Ib diamonds. Journal of Physics: Condensed Matter 2, 8567 (1990).
    19 Davies, G. The effect of nitrogen impurity on the annealing of radiation damage in diamond. Journal of Physics C: Solid State Physics 5, 2534 (1972).
    20 Jones, G., R. & P., J. Theory of aggregation of nitrogen in diamond. (2000).
    21 Huan-Cheng, C., Kowa, C. & Sun, K. Nanodiamond as a Possible Carrier of Extended Red Emission. The Astrophysical Journal Letters 639, L63 (2006).
    22 Govind, P., A.B., S. & Madhuri, S. Fishes Of Madhya Pradesh With Special Reference To Zebrafish As Model Organism In Biomedical Researches. International Research Journal of Pharmacy 3, 120-123 (2012).
    23 Bradbury, J. Small fish, big science. PLoS biology 2, E148, doi:10.1371/journal.pbio.0020148 (2004).
    24 Wixon, J. Featured organism: Danio rerio, the zebrafish. Yeast 17, 225-231, doi:10.1002/1097-0061(20000930)17:3<225::AID-YEA34>3.0.CO;2-5 (2000).
    25 Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio*). (2007).
    26 Stuart, G. W., Vielkind, J. R., McMurray, J. V. & Westerfield, M. Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression. Development 109, 577-584 (1990).
    27 Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. & Schilling, T. F. Stages of embryonic development of the zebrafish. Developmental dynamics : an official publication of the American Association of Anatomists 203, 253-310, doi:10.1002/aja.1002030302 (1995).
    28 Wolpert, L. & Tickle, C. Principles of Development. (Oxford University Press, 2010).
    29 Streisinger, G., Walker, C., Dower, N., Knauber, D. & Singer, F. Production of clones of homozygous diploid zebra fish (Brachydanio rerio). Nature 291, 293-296 (1981).
    30 III, H. W. D., Westerfield, M. & Zon, L. I. Methods in Cell Biology, Volume 59, The Zebrafish : Biology. (1998).
    31 Dodd, A., Curtis, P. M., Williams, L. C. & Love, D. R. Zebrafish: bridging the gap between development and disease. Human Molecular Genetics 9, 2443-2449, doi:10.1093/hmg/9.16.2443 (2000).
    32 Lucitt, M. B. et al. Analysis of the zebrafish proteome during embryonic development. Molecular & cellular proteomics : MCP 7, 981-994, doi:10.1074/mcp.M700382-MCP200 (2008).
    33 Pichler, F. B. et al. Chemical discovery and global gene expression analysis in zebrafish. Nat Biotechnol 21, 879-883, doi:10.1038/nbt852 (2003).
    34 Mullins, M. C., Hammerschmidt, M., Haffter, P. & Nüsslein-Volhard, C. Large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate. Current Biology 4, 189-202, doi:10.1016/s0960-9822(00)00048-8 (1994).
    35 Walker, C. & Streisinger, G. Induction of Mutations by gamma-Rays in Pregonial Germ Cells of Zebrafish Embryos. Genetics 103, 125-136 (1983).
    36 Solnica-Krezel, L., Schier, A. F. & Driever, W. Efficient recovery of ENU-induced mutations from the zebrafish germline. Genetics 136, 1401-1420 (1994).
    37 III, H. W. D., Westerfield, M. & Zon, L. I. Methods in Cell Biology, Volume 60, The Zebrafish, Genetics and Genomics. (1998).
    38 Knoll-Gellida, A. et al. Molecular phenotype of zebrafish ovarian follicle by serial analysis of gene expression and proteomic profiling, and comparison with the transcriptomes of other animals. BMC Genomics 7, 46, doi:10.1186/1471-2164-7-46 (2006).
    39 Granato, M. & Nüsslein-Volhard, C. Fishing for genes controlling development. Current Opinion in Genetics & Development 6, 461-468, doi:10.1016/s0959-437x(96)80068-2 (1996).
    40 Guyon, J. R. et al. Modeling human muscle disease in zebrafish. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1772, 205-215, doi:10.1016/j.bbadis.2006.07.003 (2007).
    41 Gestri, G., Link, B. A. & Neuhauss, S. C. F. The visual system of zebrafish and its use to model human ocular Diseases. Developmental Neurobiology 72, 302-327, doi:10.1002/dneu.20919 (2012).
    42 Whitfield, T. T. Zebrafish as a model for hearing and deafness. Journal of Neurobiology 53, 157-171, doi:10.1002/neu.10123 (2002).
    43 North, T. E. & Zon, L. I. Modeling human hematopoietic and cardiovascular diseases in zebrafish. Developmental Dynamics 228, 568-583, doi:10.1002/dvdy.10393 (2003).
    44 De Domenico, I. et al. Zebrafish as a model for defining the functional impact of mammalian ferroportin mutations. Blood 110, 3780-3783, doi:10.1182/blood-2007-07-100248 (2007).
    45 Drummond, I. A. Kidney development and disease in the zebrafish. Journal of the American Society of Nephrology : JASN 16, 299-304, doi:10.1681/ASN.2004090754 (2005).
    46 Langheinrich, U. Zebrafish: A new model on the pharmaceutical catwalk. BioEssays 25, 904-912, doi:10.1002/bies.10326 (2003).
    47 King-Heiden, T. C. et al. Quantum Dot Nanotoxicity Assessment Using the Zebrafish Embryo. Environmental Science & Technology 43, 1605-1611, doi:10.1021/es801925c (2009).
    48 Bar-Ilan, O., Albrecht, R. M., Fako, V. E. & Furgeson, D. Y. Toxicity Assessments of Multisized Gold and Silver Nanoparticles in Zebrafish Embryos. Small 5, 1897-1910, doi:10.1002/smll.200801716 (2009).
    49 Asharani, P. V., Yi Lian, W., Zhiyuan, G. & Suresh, V. Toxicity of silver nanoparticles in zebrafish models. Nanotechnology 19, 255102 (2008).
    50 Griffitt, R. J. et al. Exposure to Copper Nanoparticles Causes Gill Injury and Acute Lethality in Zebrafish (Danio rerio). Environmental Science & Technology 41, 8178-8186, doi:10.1021/es071235e (2007).
    51 Fent, K., Weisbrod, C. J., Wirth-Heller, A. & Pieles, U. Assessment of uptake and toxicity of fluorescent silica nanoparticles in zebrafish (Danio rerio) early life stages. Aquatic Toxicology 100, 218-228, doi:10.1016/j.aquatox.2010.02.019 (2010).
    52 Bai, W. et al. Toxicity of zinc oxide nanoparticles to zebrafish embryo: a physicochemical study of toxicity mechanism. Journal of Nanoparticle Research 12, 1645-1654, doi:DOI 10.1007/s11051-009-9740-9 (2010).
    53 Kane, D. A. & Kimmel, C. B. The zebrafish midblastula transition. Development 119, 447-456 (1993).
    54 Gilbert, S. F. Developmental Biology. (Sinauer Associates, 2000).
    55 Pelegri, F. Maternal factors in zebrafish development. Developmental dynamics : an official publication of the American Association of Anatomists 228, 535-554, doi:10.1002/dvdy.10390 (2003).
    56 Leung, C. F., Webb, S. E. & Miller, A. L. On the mechanism of ooplasmic segregation in single-cell zebrafish embryos. Development, Growth & Differentiation 42, 29-40, doi:10.1046/j.1440-169x.2000.00484.x (2000).
    57 Jesuthasan, S. & Strähle, U. Dynamic microtubules and specification of the zebrafish embryonic axis. Current Biology 7, 31-42, doi:10.1016/s0960-9822(06)00025-x (1997).
    58 Fernández, J., Valladares, M., Fuentes, R. & Ubilla, A. Reorganization of cytoplasm in the zebrafish oocyte and egg during early steps of ooplasmic segregation. Developmental Dynamics 235, 656-671, doi:10.1002/dvdy.20682 (2006).
    59 Fuentes, R. & Fernández, J. Ooplasmic segregation in the zebrafish zygote and early embryo: Pattern of ooplasmic movements and transport pathways. Developmental Dynamics 239, 2172-2189, doi:10.1002/dvdy.22349 (2010).
    60 Chang, Y. R. et al. Mass production and dynamic imaging of fluorescent nanodiamonds. Nature nanotechnology 3, 284-288, doi:10.1038/nnano.2008.99 (2008).
    61 Osswald, S., Yushin, G., Mochalin, V., Kucheyev, S. O. & Gogotsi, Y. Control of sp2/sp3 Carbon Ratio and Surface Chemistry of Nanodiamond Powders by Selective Oxidation in Air. Journal of the American Chemical Society 128, 11635-11642, doi:10.1021/ja063303n (2006).
    62 Kruger, A., Liang, Y., Jarre, G. & Stegk, J. Surface functionalisation of detonation diamond suitable for biological applications. Journal of Materials Chemistry 16, 2322-2328 (2006).
    63 O'Brien, G. S. et al. Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos. J Vis Exp, e1129, doi:doi:10.3791/1129 (2009).
    64 Tzeng, Y.-K. et al. Superresolution Imaging of Albumin-Conjugated Fluorescent Nanodiamonds in Cells by Stimulated Emission Depletion. Angewandte Chemie International Edition 50, 2262-2265, doi:10.1002/anie.201007215 (2011).
    65 Kimmel, C. B. & Law, R. D. Cell lineage of zebrafish blastomeres: II. Formation of the yolk syncytial layer. Developmental Biology 108, 86-93, doi:10.1016/0012-1606(85)90011-9 (1985).

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