臺灣博碩士論文加值系統

本研究主要是檢測奈米銀對再生渦蟲的毒性，在奈米銀處理後，渦蟲的運動數據蒐集並分析，我們設計一個標準化圓形軌道平台來定量及分析渦蟲的運動型態。
銀奈米粒子處理後的渦蟲在探索行為時間會增加，而經過橫切手術的渦蟲也會有增加探索時間的狀況。
觀察渦蟲在圓形軌道中的表現，對照組是以逆時針方向行進為主，橫切手術會導致順時針方向行進變多，經過銀奈米粒子處理後可以恢復以逆時針為主的行進模式。
我們發展出一套新的方法來測試渦蟲的運動行為，利用圓形軌道讓渦蟲在有限的空間中進行運動，這方法我們命名為pCOLMV(Planarian Circular Orbit LocoMotion Velocity)，預期對再生醫學的藥物開發有突破性的貢獻。

The purpose of this study is to examine the toxicity of silver nanoparticles (AgNPs) in the regenerating planarians. After the AgNPs treatments, the planarian locomotion data were collected and analyzed. A standardized circular pathways platform was developed to quantify and profiling the locomotion patterns of planarians.
The standardized circular pathways platform was designed to force planarian moving forward, however, experiments showed that the platform still allow planarian moving clockwise or counterclockwise. We defined the “looking around” behavior as explore time (ET). The explore time was increased both the AgNPs treatments groups and planarian cutting group.
The performance of planarians in circular pathways shown that the moving direction of the control group was counterclockwise , however, the planarian cutting group changed the direction clockwise. After the AgNPs treatments, the planarian moving direction was restored to counterclockwise-oriented mode .
We developed a novel approach for the locomotion testing of planarian using circular orbit in a limited space. The method, we named it “Planarian Circular Orbit LocoMotion Velocity (pCOLMV)”, can be quite useful in biomedical research, especially in the drug development for regeneration medicine.

目次
i. 口試委員審定書
ii. 授權書
iii. 誌謝辭
v. 中文摘要
vi. 英文摘要
圖目次.....................................................4
表目次.....................................................4
第一章 前言
壹、渦蟲..................................................5
貳、奈米銀粒子應用 ...............................6
一、傷口敷料 ..........................................6
二、抗菌劑 ..............................................7
參、奈米銀合成........................................7
肆、奈米銀對渦蟲的毒性.........................7
伍、研究目的............................................8
一、奈米銀對渦蟲的毒性..........................9
二、渦蟲運動速度.....................................9
三、渦蟲圓形軌道運動速度.......................9
第二章　材料與方法
壹、藥品與試劑.......................................10
一、藥品..........................................10
二、奈米銀溶液配製................................10
三、渦蟲來源...........................................10
四、渦蟲準備工作....................................11
貳、儀器..................................................13
參、實驗方法...........................................13
一、奈米銀的合成與對渦蟲的毒性...........13
二、渦蟲的預備實驗.................................16
三、運動行為資料蒐集.............................18
第三章、結果與討論
壹、渦蟲進行移動前的探索時間............................20
一、橫切手術會使探索時間增加............................20
二、奈米銀溶液處理會使探索時間增加..................20
三、奈米銀溶液處理的影響比橫切手術明顯...........21
四、奈米銀溶液處理與橫切手術有加成的效果.......21
貳、渦蟲在方向上的表現........................................22
一、渦蟲移動時以逆時針方向為主.........................22
二、橫切手術會造成渦蟲失去方向感......................23
三、奈米銀粒子可以促進渦蟲恢復方向感...............23
參、渦蟲實驗的一些新發現和設計..........................24
一、雙頭渦蟲..........................................................24
二、紅色渦蟲..........................................................24
三、3D列印圓形軌道..............................................25
第四章　結論與未來展望........................................26
第五章　參考文獻...................................................27
附錄.........................................................................33

[1] C. E. Stringer, "The means of locomotion in planarians," Proc. Natl. Acad. Sci. U. S. A., vol. 3, (12), pp. 691-692, Dec, 1917.
[2] L. Bertalanffy, O. Hoffmann-Ostenhof and O. Schreier, "A quantitative study of the toxic action of quinones on planaria gonocephala," Nature, vol. 158, (4026), pp. 948, Dec 28, 1946.
[3] W. C. Curtis, "Migration of formative cells in regeneration of planarian pharynx," Anat. Rec., vol. 94, pp. 358, Mar, 1946.
[4] E. D. Goldsmith, "Thiourea and head regeneration in planarians," Anat. Rec., vol. 96, (4), pp. 581, Dec, 1946.
[5] G. Marsh and H. W. Beams, "Electrical control of growth polarity in regenerating Dugesia tigrina," Fed. Proc., vol. 6, (1 Pt 2), pp. 163, 1947.
[6] T. Inoue, H. Hoshino, T. Yamashita, S. Shimoyama and K. Agata, "Planarian shows decision-making behavior in response to multiple stimuli by integrative brain function," Zoological Lett., vol. 1, pp. 7-014-0010-z. eCollection 2015, Feb 1, 2015.
[7] R. Langer, M. Fefferman, P. Gryska and K. Bergman, "A simple method for studying chemotaxis using sustained release of attractants from inert polymers," Can. J. Microbiol., vol. 26, (2), pp. 274-278, Feb, 1980.
[8] P. R. Mason, "Chemo-klino-kinesis in planarian food location," Anim. Behav., vol. 23, (2), pp. 460-469, May, 1975.
[9] S. D. Cross, A. A. Johnson, B. J. Gilles, L. A. Bachman, T. Inoue, K. Agata, L. Y. Marmorstein and A. D. Marmorstein, "Control of Maintenance and Regeneration of Planarian Eyes by ovo," Invest. Ophthalmol. Vis. Sci., vol. 56, (12), pp. 7604-7610, Nov, 2015.
[10] J. P. Dexter, M. B. Tamme, C. H. Lind and E. M. Collins, "On-chip immobilization of planarians for in vivo imaging," Sci. Rep., vol. 4, pp. 6388, Sep 17, 2014.
[11] T. R. Paskin, J. Jellies, J. Bacher and W. S. Beane, "Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength," PLoS One, vol. 9, (12), pp. e114708, Dec 10, 2014.
[12] G. Pescetto and D. Dettore, "Negative phototaxis and conditioning in the planarian Dugesia dorotocephala," Riv. Neurobiol., vol. 27, (2), pp. 287-295, Apr-Jun, 1982.
[13] L. Balestrini, A. Di Donfrancesco, L. Rossi, S. Marracci, M. E. Isolani, A. M. Bianucci and R. Batistoni, "The natural compound sanguinarine perturbs the regenerative capabilities of planarians," Int. J. Dev. Biol., vol. 61, (1-2), pp. 43-52, 2017.
[14] Y. Ben Khadra, M. Sugni, C. Ferrario, F. Bonasoro, A. Varela Coelho, P. Martinez and M. D. Candia Carnevali, "An integrated view of asteroid regeneration: tissues, cells and molecules," Cell Tissue Res., Mar 22, 2017.
[15] N. A. Bonar and C. P. Petersen, "Integrin suppresses neurogenesis and regulates brain tissue assembly in planarian regeneration," Development, vol. 144, (5), pp. 784-794, Mar 1, 2017.
[16] D. D. R. Brown and B. J. Pearson, "A Brain Unfixed: Unlimited Neurogenesis and Regeneration of the Adult Planarian Nervous System," Front. Neurosci., vol. 11, pp. 289, May 23, 2017.
[17] J. D. Chan, D. Zhang, X. Liu, M. Zarowiecki, M. Berriman and J. S. Marchant, "Utilizing the planarian voltage-gated ion channel transcriptome to resolve a role for a Ca2+ channel in neuromuscular function and regeneration," Biochim. Biophys. Acta, vol. 1864, (6), pp. 1036-1045, Jun, 2017.
[18] X. Chen and C. Xu, "Proteomic Analysis Reveals the Contribution of TGFbeta/Smad4 Signaling Pathway to Cell Differentiation During Planarian Tail Regeneration," Appl. Biochem. Biotechnol., vol. 182, (2), pp. 529-545, Jun, 2017.
[19] A. Y. T. Lin and B. J. Pearson, "Yorkie is required to restrict the injury responses in planarians," PLoS Genet., vol. 13, (7), pp. e1006874, Jul 7, 2017.


[20] J. Morokuma, F. Durant, K. B. Williams, J. M. Finkelstein, D. J. Blackiston, T. Clements, D. W. Reed, M. Roberts, M. Jain, K. Kimel, S. A. Trauger, B. E. Wolfe and M. Levin, "Planarian regeneration in space: Persistent anatomical, behavioral, and bacteriological changes induced by space travel," Regeneration (Oxf), vol. 4, (2), pp. 85-102, Jun 13, 2017.
[21] C. P. Petersen, "Regeneration: Organizing the Blastema in Planarians," Curr. Biol., vol. 27, (5), pp. R181-R183, Mar 6, 2017.
[22] C. Bezerra da Silva, A. Pott, S. Elifio-Esposito, L. Dalarmi, K. Fialho do Nascimento, L. Moura Burci, M. de Oliveira, J. de Fatima Gaspari Dias, S. M. Warumby Zanin, O. Gomes Miguel and M. Dallarmi Miguel, "Effect of Donepezil, Tacrine, Galantamine and Rivastigmine on Acetylcholinesterase Inhibition in Dugesia tigrina," Molecules, vol. 21, (1), pp. 53, Jan 11, 2016.
[23] M. S. Farrell, K. Gilmore, R. B. Raffa and E. A. Walker, "Behavioral characterization of serotonergic activation in the flatworm Planaria," Behav. Pharmacol., vol. 19, (3), pp. 177-182, May, 2008.
[24] R. B. Raffa, F. Cavallo and A. Capasso, "Flumazenil-sensitive dose-related physical dependence in planarians produced by two benzodiazepine and one non-benzodiazepine benzodiazepine-receptor agonists," Eur. J. Pharmacol., vol. 564, (1-3), pp. 88-93, Jun 14, 2007.
[25] R. B. Raffa, L. J. Holland and R. J. Schulingkamp, "Quantitative assessment of dopamine D2 antagonist activity using invertebrate (Planaria) locomotion as a functional endpoint," J. Pharmacol. Toxicol. Methods, vol. 45, (3), pp. 223-226, May-Jun, 2001.
[26] R. B. Raffa and A. F. Martley, "Amphetamine-induced increase in planarian locomotor activity and block by UV light," Brain Res., vol. 1031, (1), pp. 138-140, Jan 7, 2005.
[27] L. Ramakrishnan, C. Amatya, C. J. DeSaer, Z. Dalhoff and M. R. Eggerichs, "Galantamine reverses scopolamine-induced behavioral alterations in Dugesia tigrina," Invert. Neurosci., vol. 14, (2), pp. 91-101, Sep, 2014.
[28] S. M. Rawls, T. Gomez and R. B. Raffa, "An NMDA antagonist (LY 235959) attenuates abstinence-induced withdrawal of planarians following acute exposure to a cannabinoid agonist (WIN 55212-2)," Pharmacol. Biochem. Behav., vol. 86, (3), pp. 499-504, Mar, 2007.
[29] S. M. Rawls, T. Rodriguez, D. A. Baron and R. B. Raffa, "A nitric oxide synthase inhibitor (L-NAME) attenuates abstinence-induced withdrawal from both cocaine and a cannabinoid agonist (WIN 55212-2) in Planaria," Brain Res., vol. 1099, (1), pp. 82-87, Jul 12, 2006.
[30] A. C. Rodrigues, J. F. Henriques, I. Domingues, O. Golovko, V. Zlabek, C. Barata, A. M. Soares and J. L. Pestana, "Behavioural responses of freshwater planarians after short-term exposure to the insecticide chlorantraniliprole," Aquat. Toxicol., vol. 170, pp. 371-376, Jan, 2016.
[31] J. Zhang, Z. Yuan, M. Zheng, Y. Sun, Y. Wang and S. Yang, "Effects of N,N-dimethylformamide on behaviour and regeneration of planarian Dugesia japonica," Toxicol. Ind. Health, vol. 29, (8), pp. 753-760, Sep, 2013.
[32] M. Fiore, R. Moroni and L. Aloe, "Removal of the submaxillary salivary glands and infection with the trematode Schistosoma mansoni alters exploratory behavior and pain thresholds in female mice," Physiol. Behav., vol. 62, (2), pp. 399-406, Aug, 1997.
[33] W. Haas and S. Haeberlein, "Penetration of cercariae into the living human skin: Schistosoma mansoni vs. Trichobilharzia szidati," Parasitol. Res., vol. 105, (4), pp. 1061-1066, Oct, 2009.
[34] D. R. Hamilton, "Immunosuppressive effects of predator induced stress in mice with acquired immunity to Hymenolepis nana," J. Psychosom. Res., vol. 18, (3), pp. 143-153, Jun, 1974.
[35] J. Koprivnikar, C. H. Gibson and J. C. Redfern, "Infectious personalities: behavioural syndromes and disease risk in larval amphibians," Proc. Biol. Sci., vol. 279, (1733), pp. 1544-1550, Apr 22, 2012.
[36] O. R. Pagan, A. L. Rowlands and K. R. Urban, "Toxicity and behavioral effects of dimethylsulfoxide in planaria," Neurosci. Lett., vol. 407, (3), pp. 274-278, Oct 30, 2006.
[37] C. P. READ, "Soboliphyme jamesoni n. sp., a curious nematode parasite of California shrews," J. Parasitol., vol. 38, (3), pp. 203-206, Jun, 1952.
[38] T. Shomrat and M. Levin, "An automated training paradigm reveals long-term memory in planarians and its persistence through head regeneration," J. Exp. Biol., vol. 216, (Pt 20), pp. 3799-3810, Oct 15, 2013.
[39] A. M. Abdel-Mohsen, J. Jancar, R. M. Abdel-Rahman, L. Vojtek, P. Hyrsl, M. Duskova and H. Nejezchlebova, "A novel in situ silver/hyaluronan bio-nanocomposite fabrics for wound and chronic ulcer dressing: In vitro and in vivo evaluations," Int. J. Pharm., vol. 520, (1-2), pp. 241-253, Mar 30, 2017.
[40] A. Ali, I. U. Haq, J. Akhtar, M. Sher, N. Ahmed and M. Zia, "Synthesis of Ag-NPs impregnated cellulose composite material: its possible role in wound healing and photocatalysis," IET Nanobiotechnol, vol. 11, (4), pp. 477-484, Jun, 2017.
[41] Z. Di, Z. Shi, M. W. Ullah, S. Li and G. Yang, "A transparent wound dressing based on bacterial cellulose whisker and poly(2-hydroxyethyl methacrylate)," Int. J. Biol. Macromol., Jul 14, 2017.
[42] E. Gee Kee, K. Stockton, R. M. Kimble, L. Cuttle and S. M. McPhail, "Cost-effectiveness of silver dressings for paediatric partial thickness burns: An economic evaluation from a randomized controlled trial," Burns, vol. 43, (4), pp. 724-732, Jun, 2017.
[43] F. M. Helaly, S. M. El-Sawy, A. I. Hashem, A. A. Khattab and R. M. Mourad, "Synthesis and characterization of nanosilver-silicone hydrogel composites for inhibition of bacteria growth," Cont Lens Anterior Eye, vol. 40, (1), pp. 59-66, Feb, 2017.
[44] S. Huang, Z. Yu, Y. Zhang, C. Qi and S. Zhang, "In situ green synthesis of antimicrobial carboxymethyl chitosan-nanosilver hybrids with controlled silver release," Int. J. Nanomedicine, vol. 12, pp. 3181-3191, Apr 18, 2017.
[45] T. Jayaramudu, K. Varaprasad, G. M. Raghavendra, E. R. Sadiku, K. Mohana Raju and J. Amalraj, "Green synthesis of tea Ag nanocomposite hydrogels via mint leaf extraction for effective antibacterial activity," J. Biomater. Sci. Polym. Ed., pp. 1-15, Jun 22, 2017.
[46] S. Kaosaar, A. Kahru, P. Mantecca and K. Kasemets, "Profiling of the toxicity mechanisms of coated and uncoated silver nanoparticles to yeast Saccharomyces cerevisiae BY4741 using a set of its 9 single-gene deletion mutants defective in oxidative stress response, cell wall or membrane integrity and endocytosis," Toxicol. in. Vitro., vol. 35, pp. 149-162, Sep, 2016.
[47] K. W. Kwok, W. Dong, S. M. Marinakos, J. Liu, A. Chilkoti, M. R. Wiesner, M. Chernick and D. E. Hinton, "Silver nanoparticle toxicity is related to coating materials and disruption of sodium concentration regulation," Nanotoxicology, vol. 10, (9), pp. 1306-1317, Nov, 2016.
[48] J. D. Martin, T. L. Colson, V. S. Langlois and C. D. Metcalfe, "Biomarkers of exposure to nanosilver and silver accumulation in yellow perch (Perca flavescens)," Environ. Toxicol. Chem., vol. 36, (5), pp. 1211-1220, May, 2017.
[49] D. Sundeep, T. Vijaya Kumar, P. S. S. Rao, R. V. S. S. N. Ravikumar and A. Gopala Krishna, "Green synthesis and characterization of Ag nanoparticles from Mangifera indica leaves for dental restoration and antibacterial applications," Prog. Biomater., vol. 6, (1-2), pp. 57-66, May, 2017.
[50] T. BETCHAKU, "A copper sulfate-silver nitrate method for nerve fibers of planarians," Stain Technol., vol. 35, pp. 215-218, Jul, 1960.
[51] I. D. Bowen, T. A. Ryder and C. Winters, "The distribution of oxidizable mucosubstances and polysaccharides in the planarian Polycelis tenuis Iijima," Cell Tissue Res., vol. 161, (2), pp. 263-275, Aug 18, 1975.
[52] L. L. DiCiaula, G. L. Foley and D. J. Schaeffer, "Fixation and staining of planaria for histological study," Biotech. Histochem., vol. 70, (3), pp. 119-123, May, 1995.
[53] I. Hori, "Localization of newly synthesized precursors of basal lamina in the regenerating planarian as revealed by autoradiography," Tissue Cell, vol. 12, (3), pp. 513-521, 1980.
[54] L. Kustov, K. Tiras, S. Al-Abed, N. Golovina and M. Ananyan, "Estimation of the toxicity of silver nanoparticles by using planarian flatworms," Altern. Lab. Anim., vol. 42, (1), pp. 51-58, Mar, 2014.