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系統識別號 U0026-0907201815280100
論文名稱(中文) 藉由模板引導自組裝調控乙型類澱粉蛋白的奈米結構
論文名稱(英文) Regulation of Aβ Aggregate Morphology by Template-Guiding Self-Assembly
校院名稱 成功大學
系所名稱(中) 化學系
系所名稱(英) Department of Chemistry
學年度 106
學期 2
出版年 107
研究生(中文) 陳郁諠
研究生(英文) Yu-Syuan Chen
學號 L36054168
學位類別 碩士
語文別 中文
論文頁數 68頁
口試委員 指導教授-李介仁
口試委員-詹揚翔
口試委員-廖尉斯
中文關鍵字 乙型類澱粉蛋白  自組裝  環形奈米結構 
英文關鍵字 amyloid beta peptide  self-assembly  ring-shaped nanostructures 
學科別分類
中文摘要 前類澱粉蛋白質(amyloid precursor protein, APP)為膜蛋白中的嵌入蛋白,主要集中於腦內神經細胞上,當其經β-分泌酵素與γ-分泌酵素依序水解後即會形成乙型類澱粉蛋白(amyloid beta peptide, Aβ)。乙型類澱粉蛋白錯誤摺疊後會異常聚集並自組裝形成寡聚物和纖維,最後形成斑塊並導致死亡,此為阿茲海默症的主因。然而目前尚未得知乙型類澱粉蛋白以何種自組裝形態與細胞表面作用,所以為了研究此蛋白自組裝聚集物結構對細胞的影響,在此我們採用兩種策略製備不同的奈米結構:(1)溶液中自然自組裝(2)模板引導自組裝。在策略一溶液自然自組裝中,藉由改變乙型類澱粉蛋白儲存時間、濃度、溶液pH值和培育方式等,我們成功改變乙型類澱粉蛋白寡聚物與纖維的尺寸、形貌,但其結構僅局限於球狀寡聚物與線狀纖維,故採用策略二藉由模板引導改變其自組裝的聚集物結構。在策略二我們使用緊密堆積的奈米球作為模板再結合奈米流體的概念,利用毛細作用力引導乙型類澱粉蛋白到模板的特定位置進行自組裝,藉由改變乾燥時間、奈米球模板尺寸、乙型類澱粉蛋白溶液與奈米球溶液體積比例等來調控,最後成功製備出不同於球狀寡聚物與線狀纖維的環形奈米結構。最後利用製備出的各式乙型類澱粉蛋白奈米結構進行細胞實驗,用以觀察不同自組裝形貌對細胞的影響。
英文摘要 Amyloid precursor protein (APP) is one of integral membrane protein that concentrates on nerve cells in the brain. APP is sequentially cleaved by β-secretase and γ-secretase, then to produce amyloid beta peptide (Aβ). Aβ peptide misfolding usually results in abnormal aggregation to form oligomers and fibrils, which are toxic to cause cell death and Alzheimer's disease (AD). However, understanding which type of Aβ aggregates interacts with the cell membrane still remains challenging. To study the influence of Aβ aggregate types on the cell membrane, we adopt two strategies to produce Aβ nanostructures: (1) solution-based natural assembly and (2) template-guiding self-assembly. With changing Aβ concentration, adsorption time, solution pH and incubation approach, we observed Aβ oligomers and fibrils randomly adsorbed on surface. Therefore, the close-packed nanoparticles serve as structural templates to guide deposition of Aβ on surface. By controlling drying time, template size and Aβ-to-particle ratio, we successfully fabricated ring-shaped Aβ nanostructures on surface. The morphology of Aβ nanorings remains uniform and the size are tunable with template particle diameter. Further, those various Aβ nanostructures will be used to investigate the impact of the shape and size of nanoscale Aβ aggregates on cell membrane.
論文目次 中文摘要 I
英文摘要 II
目錄 VII
表目錄 IX
圖目錄 IX
第一章、 緒論 1
一、 前言 1
二、 阿茲海默症 1
2.1 類澱粉沉積症 1
2.2 乙型類澱粉蛋白之自組裝與其毒性 2
三、 蛋白質奈米結構製程 4
四、 奈米流體 7
五、 環形水膜與環間水橋 7
六、 原子力顯微鏡 8
第二章、 研究乙型類澱粉蛋白在不同環境下自組裝之形貌變化 11
一、 實驗目的 11
二、 實驗部分 12
2.1 實驗試藥 12
2.2 儀器裝置 12
2.3 實驗步驟 13
三、 結果與討論 16
3.1乙型類澱粉蛋白儲存時間對其在溶液中自組裝的影響 16
3.2乙型類澱粉蛋白濃度對其在溶液中自組裝的影響 17
3.3乙型類澱粉蛋白吸附時間對其在雲母片上自組裝的影響 18
3.4乙型類澱粉蛋白溶液pH值對其在溶液中自組裝的影響 20
3.5乙型類澱粉蛋白不同培育方式對其自組裝的影響 23
四、 結論 28
第三章、 利用奈米球模板控制乙型類澱粉蛋白自組裝形成均一奈米結構 29
一、 實驗目的 29
二、 實驗部分 30
2.1 實驗試藥 30
2.2 儀器裝置 31
2.3 實驗策略 31
2.4 實驗步驟 32
三、 結果與討論 38
3.1各式尺寸的奈米球模板 38
3.2牛血清白蛋白環形奈米結構 39
3.3分析給予奈米球模板時機對乙型類澱粉蛋白環形奈米結構的影響 41
3.4分析乙型類澱粉蛋白不同培育方式對乙型類澱粉蛋白環形奈米結構的影響 42
3.5分析奈米流體蒸發時間對乙型類澱粉蛋白環形奈米結構的影響 44
3.6分析奈米球直徑對乙型類澱粉蛋白環形奈米結構的影響 47
3.7分析調控溶液中奈米球與乙型類澱粉蛋白比例對乙型類澱粉蛋白環形奈米結構的影響 52
3.8測試乙型類澱粉蛋白環形奈米結構的穩定性 57
3.9分析乙型類澱粉蛋白環形奈米結構的細胞實驗 59
3.10 分析縮短乙型類澱粉蛋白儲存時間對乙型類澱粉蛋白環形奈米結構的影響 60
四、 結論 63
參考文獻 64

參考文獻 [1] Finder, V. H.; Glockshuber, R., Amyloid-β aggregation. Neurodegenerative Diseases
2007, 4 (1), 13-27.
[2] Cerf, E.; Sarroukh, R.; Tamamizu-Kato, S.; Breydo, L.; Derclaye, S.; Dufrêne, Y. F.; Narayanaswami, V.; Goormaghtigh, E.; Ruysschaert, J.-M.; Raussens, V., Antiparallel β-sheet: a signature structure of the oligomeric amyloid β-peptide. Biochemical Journal 2009, 421 (3), 415-423.
[3] Knowles, T. P.; Buehler, M. J., Nanomechanics of functional and pathological amyloid materials. Nature Nanotechnology 2011, 6 (8), 469-479.
[4] Rochet, J.-C.; Lansbury Jr, P. T., Amyloid fibrillogenesis: themes and variations. Current Opinion in Structural Biology 2000, 10 (1), 60-68.
[5] Sipe, J. D.; Benson, M. D.; Buxbaum, J. N.; Ikeda, S.-i.; Merlini, G.; Saraiva, M. J.; Westermark, P., Nomenclature 2014: amyloid fibril proteins and clinical classification of the amyloidosis. Amyloid 2014, 21 (4), 221-224.
[6] Organization, W. H., Dementia cases set to triple by 2050 but still largely ignored. Neurosciences 2012, 17 (3), 296-269.
[7] WHO: dementia. http://www.who.int/en/news-room/fact-sheets/detail/dementia.
[8] The top 10 causes of death 2015. http://origin.who.int/mediacentre/factsheets/fs310/en/.
[9] The top 10 causes of death 2016. http://www.who.int/en/news-room/fact-sheets/detail/the-top-10-causes-of-death.
[10] Priller, C.; Bauer, T.; Mitteregger, G.; Krebs, B.; Kretzschmar, H. A.; Herms, J., Synapse formation and function is modulated by the amyloid precursor protein. Journal of Neuroscience 2006, 26 (27), 7212-7221.
[11] O'Brien, R. J.; Wong, P. C., Amyloid precursor protein processing and Alzheimer's disease. Annual Review of Neuroscience 2011, 34, 185-204.
[12] Zhang, C.; Tanzi, R. E., Natural modulators of amyloid-beta precursor protein processing. Current Alzheimer Research 2012.
[13] Huang, L.; Su, X.; Federoff, H. J., Single-chain fragment variable passive immunotherapies for neurodegenerative diseases. International Journal of Molecular Sciences 2013, 14 (9), 19109-19127.
[14] Wang, Q.; Zhao, J.; Yu, X.; Zhao, C.; Li, L.; Zheng, J., Alzheimer Aβ1− 42 Monomer Adsorbed on the Self-Assembled Monolayers. Langmuir 2010, 26 (15), 12722-12732.
[15] Selkoe, D. J., Alzheimer's disease: genes, proteins, and therapy. Physiological Reviews 2001, 81 (2), 741-766.
[16] Kraig, R. P.; Pulsinelli, W. A.; Plum, F., Heterogeneous Distribution of Hydrogen and Bicarbonate Ions During Complete Brain Cischemia. Progress in Brain Research 1985, 63, 155-166.
[17] Malakooti, N.; Pritchard, M. A.; Adlard, P. A.; Finkelstein, D. I., Role of metal ions in the cognitive decline of Down syndrome. Frontiers in Aging Neuroscience 2014, 6, 136.
[18] Hoff, J. D.; Cheng, L.-J.; Meyhöfer, E.; Guo, L. J.; Hunt, A. J., Nanoscale protein patterning by imprint lithography. Nano Letters 2004, 4 (5), 853-857.
[19] Falconnet, D.; Pasqui, D.; Park, S.; Eckert, R.; Schift, H.; Gobrecht, J.; Barbucci, R.; Textor, M., A novel approach to produce protein nanopatterns by combining nanoimprint lithography and molecular self-assembly. Nano Letters 2004, 4 (10), 1909-1914.
[20] Irvine, E. J.; Hernandez-Santana, A.; Faulds, K.; Graham, D., Fabricating protein immunoassay arrays on nitrocellulose using dip-pen lithography techniques. Analyst 2011, 136 (14), 2925-2930.
[21] Kim, S. M.; Suh, K. Y., Fabrication of biological arrays by unconventional lithographic methods. Frontiers in Bioscience S 2009, 1, 406-419.
[22] Lange, S. A.; Benes, V.; Kern, D. P.; Hörber, J. H.; Bernard, A., Microcontact printing of DNA molecules. Analytical Chemistry 2004, 76 (6), 1641-1647.
[23] Renault, J.; Bernard, A.; Bietsch, A.; Michel, B.; Bosshard, H.; Delamarche, E.; Kreiter, M.; Hecht, B.; Wild, U., Fabricating arrays of single protein molecules on glass using microcontact printing. The Journal of Physical Chemistry B 2003, 107 (3), 703-711.
[24] Englade-Franklin, L. E.; Saner, C. K.; Garno, J. C., Spatially selective surface platforms for binding fibrinogen prepared by particle lithography with organosilanes. Interface Focus 2013, 3 (3), 20120102.
[25] Yu, W.; France, D. M.; Choi, S. U.; Routbort, J. L. Review and assessment of nanofluid technology for transportation and other applications; Argonne National Laboratory (ANL): 2007.
[26] Bhardwaj, R.; Fang, X.; Somasundaran, P.; Attinger, D., Self-assembly of colloidal particles from evaporating droplets: role of DLVO interactions and proposition of a phase diagram. Langmuir 2010, 26 (11), 7833-7842.
[27] Denkov, N.; Velev, O.; Kralchevski, P.; Ivanov, I.; Yoshimura, H.; Nagayama, K., Mechanism of formation of two-dimensional crystals from latex particles on substrates. Langmuir 1992, 8 (12), 3183-3190.
[28] Bigioni, T. P.; Lin, X.-M.; Nguyen, T. T.; Corwin, E. I.; Witten, T. A.; Jaeger, H. M., Kinetically driven self assembly of highly ordered nanoparticle monolayers. Nature Materials 2006, 5 (4), 265-270.
[29] Yang, L.; Hu, J.; Bai, K., Capillary and van der Waals force between microparticles with different sizes in humid air. Journal of Adhesion Science and Technology 2016, 30 (5), 566-578.
[30] Leroch, S.; Wendland, M., Influence of capillary bridge formation onto the silica nanoparticle interaction studied by grand canonical Monte Carlo simulations. Langmuir 2013, 29 (40), 12410-12420.
[31] Dörmann, M.; Schmid, H.-J., Simulation of capillary bridges between nanoscale particles. Langmuir 2014, 30 (4), 1055-1062.
[32] Emory college of arts and sciences: atomic force microscopy http://www.physics.emory.edu/faculty/finzi/research/afm.html.
[33] IUPUI: gneral introduction to atomic force microscopy. http://www.iupui.edu/~bbml/afmintro.html.
[34] Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D. B., Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways. PNAS 2003, 100 (1), 330-335.
[35] Ha, C.; Park, C. B., Ex situ atomic force microscopy analysis of β-amyloid self-assembly and deposition on a synthetic template. Langmuir 2006, 22 (16), 6977-6985.
[36] Moores, B.; Drolle, E.; Attwood, S. J.; Simons, J.; Leonenko, Z., Effect of surfaces on amyloid fibril formation. PLoS One 2011, 6 (10), e25954.
[37] Broersen, K.; Jonckheere, W.; Rozenski, J.; Vandersteen, A.; Pauwels, K.; Pastore, A.; Rousseau, F.; Schymkowitz, J., A standardized and biocompatible preparation of aggregate-free amyloid beta peptide for biophysical and biological studies of Alzheimer's disease. Protein Engineering, Design and Selection 2011, 24 (9), 743-750.
[38] Bin, Y.; Li, X.; He, Y.; Chen, S.; Xiang, J., Amyloid-β peptide (1–42) aggregation induced by copper ions under acidic conditions. Acta Biochim Biophys Sin 2013, 45 (7), 570-577.
[39] Lin, Y.-C.; Petersson, E. J.; Fakhraai, Z., Surface effects mediate self-assembly of amyloid-β peptides. ACS nano 2014, 8 (10), 10178-10186.
[40] Ryan, T. M.; Caine, J.; Mertens, H. D.; Kirby, N.; Nigro, J.; Breheney, K.; Waddington, L. J.; Streltsov, V. A.; Curtain, C.; Masters, C. L., Ammonium hydroxide treatment of Aβ produces an aggregate free solution suitable for biophysical and cell culture characterization. PeerJ 2013, 1, e73.
[41] Senden, T. J.; Ducker, W. A., Surface roughness of plasma-treated mica. Langmuir 1992, 8 (2), 733-735.
[42] Dubnovitsky, A.; Sandberg, A.; Rahman, M. M.; Benilova, I.; Lendel, C.; Härd, T., Amyloid-β protofibrils: size, morphology and synaptotoxicity of an engineered mimic. PloS one 2013, 8 (7), e66101.
[43] Khurana, R.; Coleman, C.; Ionescu-Zanetti, C.; Carter, S. A.; Krishna, V.; Grover, R. K.; Roy, R.; Singh, S., Mechanism of thioflavin T binding to amyloid fibrils. Journal of Structural Biology 2005, 151 (3), 229-238.
[44] Biancalana, M.; Koide, S., Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochimica et Biophysica Acta 2010, 1804 (7), 1405-1412.
[45] Xue, C.; Lin, T. Y.; Chang, D.; Guo, Z., Thioflavin T as an amyloid dye: fibril quantification, optimal concentration and effect on aggregation. Royal Society Open Science 2017, 4 (1), 160696.
[46] Maskevich, A. A.; Stsiapura, V. I.; Kuzmitsky, V. A.; Kuznetsova, I. M.; Povarova, O. I.; Uversky, V. N.; Turoverov, K. K., Spectral properties of thioflavin T in solvents with different dielectric properties and in a fibril-incorporated form. Journal of Proteome Research 2007, 6 (4), 1392-1401.
[47] Hackl, E. V.; Darkwah, J.; Smith, G.; Ermolina, I., Effect of acidic and basic pH on Thioflavin T absorbance and fluorescence. European Biophysics Journal 2015, 44 (4), 249-261.
[48] Levine, H., Thioflavine T interaction with synthetic Alzheimer's disease β‐amyloid peptides: Detection of amyloid aggregation in solution. Protein Science 1993, 2 (3), 404-410.
[49] Groenning, M., Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils—current status. Journal of Chemical Biology 2010, 3 (1), 1-18.
[50] Bode, D. C.; Baker, M. D.; Viles, J. H., Ion Channel Formation by Amyloid-β42 Oligomers but not Amyloid-β40 in Cellular Membranes. Journal of Biological Chemistry 2017, 292 (4), 1404-1413.
[51] Lin, H.; Bhatia, R.; Lal, R., Amyloid β protein forms ion channels: implications for Alzheimer’s disease pathophysiology. The FASEB Journal 2001, 15 (13), 2433-2444.
[52] Jang, H.; Arce, F. T.; Ramachandran, S.; Capone, R.; Azimova, R.; Kagan, B. L.; Nussinov, R.; Lal, R., Truncated β-amyloid peptide channels provide an alternative mechanism for Alzheimer’s disease and Down syndrome. PNAS 2010, 107 (14), 6538-6543.
[53] Quist, A.; Doudevski, I.; Lin, H.; Azimova, R.; Ng, D.; Frangione, B.; Kagan, B.; Ghiso, J.; Lal, R., Amyloid ion channels: a common structural link for protein-misfolding disease. PNAS 2005, 102 (30), 10427-10432.
[54] Mesquida, P.; Blanco, E. M.; McKendry, R. A., Patterning amyloid peptide fibrils by AFM charge writing. Langmuir 2006, 22 (22), 9089-9091.
[55] Hulteen, J. C.; Van Duyne, R. P., Nanosphere lithography: A materials general fabrication process for periodic particle array surfaces. Journal of Vacuum Science and Technology A 1995, 13 (3), 1553-1558.
[56] Ngunjiri, J. N.; Daniels, S. L.; Li, J.-R.; Serem, W. K.; Garno, J. C., Controlling the surface coverage and arrangement of proteins using particle lithography. Nanomedicine 2008, 3 (4), 529-541.
[57] Li, J.-R.; Henry, G. C.; Garno, J. C., Fabrication of nanopatterned films of bovine serum albumin and staphylococcal protein A using latex particle lithography. Analyst 2006, 131 (2), 244-250.
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