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系統識別號 U0026-1507201721265400
論文名稱(中文) 秀麗隱桿線蟲多面向研究之初期整合微晶片開發
論文名稱(英文) Preliminary Development of an Integrated Microchip for Multipurpose Caenorhabditis elegans Studies
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 105
學期 2
出版年 106
研究生(中文) 莊婉愉
研究生(英文) Wan-Yu Chuang
學號 P86034179
學位類別 碩士
語文別 英文
論文頁數 57頁
口試委員 指導教授-莊漢聲
口試委員-陳昌熙
口試委員-陳嘉元
口試委員-邱文泰
中文關鍵字 秀麗隱桿線蟲  運動  紫外光  可逆式固定  水凝膠PF-127  光電裝置  癌症篩檢  微流道晶片 
英文關鍵字 Caenorhabditis (C.) elegans  Exercise  Ultraviolet (UV) light  Reversible immobilization  Pluronic (P) F-127  Optoelectric device  Cancer screening  Microchip 
學科別分類
中文摘要 人類壽命的延長導致罹患退化性疾病與癌症的人口大幅上升。如何延緩老化以減少病症的發生或提供較好的預後便是一項重要的議題。由於多細胞生物秀麗隱桿線蟲的基因與人類高度相似,因此常被廣泛用於各研究領域,例如神經學、發育學及藥物學。然而截至目前為止相關研究仍仰賴繁瑣的人為操作,大為降低研究產率及效率。本研究發展一套整合微晶片,針對固定觀測、運動以及癌症篩檢三個部分,透過線蟲初步確立微晶片之功能分析。期望未來直接於晶片上執行線蟲培養與多面向研究,提升操作的效率與便利性。
不論線蟲的顯微手術、螢光造影或生理特徵的觀察,固定是道不可或缺的程序,因此我們將此功能整合在微晶片上。本研究以光電裝置搭配20% w/v水凝膠PF-127固定線蟲。經過探討發現以最佳化條件同時施加雷射及電場,線蟲能在裝置開啟後4秒被固定,而於關閉迴路後1秒恢復正常游動。改變雷射大小及照射區域還能改變成膠的範圍與位置,達到線蟲的局部或全身固定。根據評估線蟲每次置於PF-127應小於3小時,而PF-127因為具有熱可逆的特性,所以線蟲不會因為固定步驟而犧牲,提升了實驗的一致性。此技術除了固定線蟲未來也能應用在其他微生物上。
運動能使生物個體更有活力並減少退化性疾病的發生。由於線蟲對短波長光線敏感,本研究捨棄以往的電趨方式,改為以紫外光(364-373 nm)刺激方法促使線蟲運動,大幅簡化複雜的流道設計。將線蟲放入液滴中4分鐘後以每2分鐘刺激5秒的頻率照射紫外光4次,能有效促使線蟲每日持續游動20分鐘以達到運動的效果。隨著人類壽命延長,癌症也是另一項棘手的問題。於癌細胞培養數日後,我們將線蟲放入濾除癌細胞之培養液觀察線蟲游動變化。不論置於子宮頸癌或大腸癌細胞的培養液,線蟲的運動型態不再以向前游動為主,擺動頻率及單位輸出功率亦有所改變。這可能是由於線蟲高度發展的化學偵測系統造成的結果。因此透過線蟲做為癌症初步篩檢工具將有助患者早期發現與治療。
透過各個功能初期的研究與分析,未來此整合晶片將可運用在癌症篩檢,或是運動和抗氧化藥物與老化之間的關聯發展,期望最終成果對退化疾病及癌症治療相關研究上有所貢獻,改善人類老年的生活品質。
英文摘要 Despite prolonging the life of humans, the number of people suffering from neurodegenerative diseases and cancer increases significantly. Therefore, postponing aging to decrease the incidence of diseases and provide better prognosis is important. Given its high similarity with humans in terms of genes, the multicellular organism Caenorhabditis elegans is the simplest model animal widely used in many research fields, such as neurology, genetic engineering, developmental biology, and pharmaceutics. However, research on the nematode still requires painstaking operations, thus reducing the throughput and efficiency. To address the problem, an integrated microchip was proposed in this study as a solution. This study is divided into three parts, namely, worm exercise, cancer screening, and immobilization.
Regardless of whether microsurgery, fluorescent imaging, or physiological observation is used in this tiny organism, immobilization is an essential step. However, minimal work has been performed. Hence, immobilization is an essential function fabricated on microchips. An immobilization technique based on the combined use of an optoelectric device and a 20% w/v thermos-reversible hydrogel solution, Pluronic F-127, was developed first. Second, the optoelectric device was coated with a photoconductive layer to allow the local circuit channels to be rapidly switched by optical illumination. After simultaneously applying light and electric fields under optimal conditions, the hydrogel reached gelation within 4 s, and the immobilized C. elegans appeared to resume its full locomotion within 1 s after the light was switched off. The gelation region and location could be manipulated by changing the laser size and illuminated region. According to the assessments, worms should not be exposed to the hydrogel environment for more than 3 h. Given the thermo-reversible property of PF-127, the sample was also conserved in the entire experiment. Aside from C. elegans, this technique can be applied to other microorganisms.
Exercise not only makes an organism more energetic during the aging process; it can also postpone the occurrence of degenerative diseases. Short-wavelength light elicits a photophobic, movement-reversal response from C. elegans. Rather than using the electrotaxis method, ultraviolet light was introduced to keep C. elegans swimming to achieve the effect of exercise. Therefore, the design of the channel was simplified significantly. After a 4 min delay in the droplet, worms that received stimuli of 5 s UV light every 2 min for 8 min accomplished 20 min of continuous exercise. Cancer is another issue linked with human aging. We placed C. elegans in the supernatant of Caco-2 and HeLa cell line culture medium separately to examine the biomechanical performance of C. elegans. Worms in the cancer cell culture medium tended to swim oddly instead of the normal forward movement. The body bend frequency and unit kinetic power decreased significantly. The results contribute to the highly developed chemosensory system of C. elegans. Therefore, using C. elegans as a sensor for cancer screening might help in the early diagnosis and successful treatment of the disease.
The proposed integrated chip can be subsequently performed for multipurpose analyses. Thus, the mechanism by which C. elegans reacts to the secretion of cancer cells and its relationship with exercise, antioxidants, and aging must be determined. The results are expected to provide information on the treatment of degenerative diseases and cancer in higher animal forms.
論文目次 摘要 I
ABSTRACT II
誌謝 IV
CONTENT V
LIST OF TABLES VIII
LIST OF FIGURES IX
CHAPTER 1 INTRODUCTION 1
1.1 Background 1
1.2 Motivation and Purpose 3
1.3 Caenorhabditis (C.) elegans 4
1.4 Pluronic F-127 5
CHAPTER 2 MATERIALS AND METHODS 7
2.1 Flow Chart of Research Process 7
2.2 Basics of C. elegans 8
2.2.1 Strains and Growth Condition of C. elegans 8
2.2.1.1 Wild-Type Worm 8
2.2.1.2 Transgenic Worm 8
2.2.2 C. elegans Culture Media Protocols 9
2.2.3 Age-Synchronized Method 9
2.2.4 Growth Assay 10
2.2.5 Progeny Assay 10
2.2.6 Lifespan Assay 10
2.2.7 Statistical Analysis 11
2.3 Integrated Microchip 11
2.3.1 Fabrication of the Integrated Microchip 11
2.4 Worm Immobilization 12
2.4.1 Preparation of PF-127 12
2.4.2 Immobilization Device and Operation 13
2.4.3 Image Processing and Analysis 14
2.4.3.1 Kymogram of Body Curvature 15
2.4.3.2 Gait Correlation Diagram 16
2.4.3.3 Trajectories 17
2.4.4 Water Bathing Technique 18
2.4.5 Stress Response Assay 18
2.4.6 Temperature Measurement 19
2.5 Exercise 20
2.5.1 Photophobic Behavior of C. elegans 20
2.5.2 Ultraviolet Light Stimulation 21
2.5.3 Measurement of the Worm’s Body Bends 21
2.6 Cancer Screening 22
2.6.1 Locomotive Gaits of C. elegans 22
2.6.2 Preparation of Culture Media from Cancer Cells 23
2.6.3 Behavior Analysis of C. elegans 24
2.6.4 Measurement of Kinetic Power 24
CHAPTER 3 RESULTS AND DISCUSSION 26
3.1 Integrated Microchip 26
3.1.1 Operation 26
3.2 Worm Immobilization 27
3.2.1 Effect of Long Exposure to PF-127 27
3.2.2 Exposure Time Evaluation 27
3.2.3 Assessment of Worm Immobilization 30
3.2.4 Addressable Immobilization 32
3.2.5 Long-term Imaging of Worm Senescence Process 34
3.3 Exercise 36
3.3.1 Effect of the Stimulation by Ultraviolet Light 37
3.3.2 Reproducibility of Photophobic Behavior 38
3.4 Cancer Screening 40
3.4.1 Gait Evaluation 40
3.4.2 Kinetic Power 42
CHAPTER 4 CONCLUSION 44
CHAPTER 5 PROSPECTS 46
REFERENCES 47
APPENDIX 55
參考文獻 [1] N. Kim, C. M. Dempsey, J. V. Zoval, J.-Y. Sze, and M. J. Madou, “Automated microfluidic compact disc (CD) cultivation system of Caenorhabditis elegans,” Sensors and Actuators B: Chemical, vol. 122, no. 2, pp. 511-518, March, 2007.
[2] N. Chronis, “Worm chips: microtools for C. elegans biology,” Lab on a Chip, vol. 10, no. 4, pp. 432-437, December, 2009.
[3] N. A. Bakhtina, and J. G. Korvink, “Microfluidic laboratories for C. elegans enhance fundamental studies in biology,” Rsc Advances, vol. 4, no. 9, pp. 4691-4709, November, 2013.
[4] K. Chung, M. M. Crane, and H. Lu, “Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans,” Nature Methods, vol. 5, no. 7, pp. 637-643, June, 2008.
[5] S. E. Hulme, S. S. Shevkoplyas, A. P. McGuigan, J. Apfeld, W. Fontana, and G. M. Whitesides, “Lifespan-on-a-chip: microfluidic chambers for performing lifelong observation of C. elegans,” Lab on a Chip, vol. 10, no. 5, pp. 589-597, December, 2009.
[6] H. Wen, W. Shi, and J. Qin, “Multiparameter evaluation of the longevity in C. elegans under stress using an integrated microfluidic device,” Biomedical Microdevices, vol. 14, no. 4, pp. 721-728, August, 2012.
[7] R. Kerr, V. Lev-Ram, G. Baird, P. Vincent, R. Y. Tsien, and W. R. Schafer, “Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans,” Neuron, vol. 26, no. 3, pp. 583-594, June, 2000.
[8] D. L. Riddle, T. Blumenthal, B. J. Meyer, and J. R. Priess, “1 Introduction to C. elegans,” Cold Spring Harbor Monograph Archive, vol. 33, pp. 1-22, 1997.
[9] S. E. Hulme, S. S. Shevkoplyas, J. Apfeld, W. Fontana, and G. M. Whitesides, “A microfabricated array of clamps for immobilizing and imaging C. elegans,” Lab on a Chip, vol. 7, no. 11, pp. 1515-1523, August, 2007.
[10] H.-S. Chuang, H.-Y. Chen, C.-S. Chen, and W.-T. Chiu, “Immobilization of the nematode Caenorhabditis elegans with addressable light-induced heat knockdown (ALINK),” Lab on a Chip, vol. 13, no. 15, pp. 2980-2989, April, 2013.
[11] J. Krajniak, and H. Lu, “Long-term high-resolution imaging and culture of C. elegans in chip-gel hybrid microfluidic device for developmental studies,” Lab on a Chip, vol. 10, no. 14, pp. 1862-1868, May, 2010.
[12] G. Aubry, M. Zhan, and H. Lu, “Hydrogel-droplet microfluidic platform for high-resolution imaging and sorting of early larval Caenorhabditis elegans,” Lab on a Chip, vol. 15, no. 6, pp. 1424-1431, January, 2015.
[13] J. Krajniak, Y. Hao, H. Y. Mak, and H. Lu, “CLIP–continuous live imaging platform for direct observation of C. elegans physiological processes,” Lab on a Chip, vol. 13, no. 15, pp. 2963-2971, April, 2013.
[14] H. Hwang, J. Krajniak, Y. Matsunaga, G. M. Benian, and H. Lu, “On-demand optical immobilization of Caenorhabditis elegans for high-resolution imaging and microinjection,” Lab on a Chip, vol. 14, no. 18, pp. 3498-3501, July, 2014.
[15] K. S. Nair, “Aging muscle,” The American Journal of Clinical Nutrition, vol. 81, no. 5, pp. 953-963, May, 2005.
[16] R. S. Mazzeo, P. Cavanagh, W. J. Evans, M. Fiatarone, J. Hagberg, E. McAuley, and J. Startzell, “Exercise and physical activity for older adults,” Medicine and Science in Sports and Exercise, vol. 30, no. 6, pp. 992-1008, June, 1998.
[17] A. Navarro, C. Gomez, J. M. López-Cepero, and A. Boveris, “Beneficial effects of moderate exercise on mice aging: survival, behavior, oxidative stress, and mitochondrial electron transfer,” American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, vol. 286, no. 3, pp. R505-R511, March, 2004.
[18] H.-S. Chuang, W.-J. Kuo, C.-L. Lee, I.-H. Chu, and C.-S. Chen, “Exercise in an electrotactic flow chamber ameliorates age-related degeneration in Caenorhabditis elegans.,” Scientific Reports, vol. 6, pp. 28064 -28064, June, 2016.
[19] B. Stewart, and C. P. Wild,“World cancer report 2014,” World, April, 2016.
[20] K. Matsumura, M. Opiekun, H. Oka, A. Vachani, S. M. Albelda, K. Yamazaki, and G. K. Beauchamp, “Urinary volatile compounds as biomarkers for lung cancer: a proof of principle study using odor signatures in mouse models of lung cancer,” PLoS One, vol. 5, no. 1, pp. e8819, January, 2010.
[21] C. I. Bargmann, “Chemosensation in C. elegans,” Wormbook, October, 2006.
[22] H. Sonoda, S. Kohnoe, T. Yamazato, Y. Satoh, G. Morizono, K. Shikata, M. Morita, A. Watanabe, M. Morita, and Y. Kakeji, “Colorectal cancer screening with odour material by canine scent detection,” Gut, vol. 60, no. 6, pp. 814 -819, January, 2011.
[23] M. McCulloch, T. Jezierski, M. Broffman, A. Hubbard, K. Turner, and T. Janecki, “Diagnostic accuracy of canine scent detection in early-and late-stage lung and breast cancers,” Integrative Cancer Therapies, vol. 5, no. 1, pp. 30-39, March, 2006.
[24] T. Hirotsu, H. Sonoda, T. Uozumi, Y. Shinden, K. Mimori, Y. Maehara, N. Ueda, and M. Hamakawa, “A highly accurate inclusive cancer screening test using Caenorhabditis elegans scent detection,” PloS One, vol. 10, no. 3, pp. e0118699, March, 2015.
[25] T. Kaletta, and M. O. Hengartner, “Finding function in novel targets: C. elegans as a model organism,” Nature Reviews Drug Discovery, vol. 5, no. 5, pp. 387-399, May, 2006.
[26] S. Brenner, “The genetics of Caenorhabditis elegans,” Genetics, vol. 77, no. 1, pp. 71-94, May, 1974.
[27] D. J. Dickinson, J. D. Ward, D. J. Reiner, and B. Goldstein, “Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination,” Nature Methods, vol. 10, no. 10, pp. 1028-1034, September, 2013.
[28] V. Robert, and J. L. Bessereau, “Targeted engineering of the Caenorhabditis elegans genome following Mos1‐triggered chromosomal breaks,” The EMBO Journal, vol. 26, no. 1, pp. 170-183, July, 2006.
[29] X. Zhao, F. Xu, L. Tang, W. Du, X. Feng, and B.-F. Liu, “Microfluidic chip-based C. elegans microinjection system for investigating cell–cell communication in vivo,” Biosensors and Bioelectronics, vol. 50, pp. 28-34, December, 2013.
[30] M. M. Crane, J. N. Stirman, C.-Y. Ou, P. T. Kurshan, J. M. Rehg, K. Shen, and H. Lu, “Autonomous screening of C. elegans identifies genes implicated in synaptogenesis,” Nature Methods, vol. 9, no. 10, pp. 977-980, August, 2012.
[31] L. Ségalat, “Invertebrate animal models of diseases as screening tools in drug discovery,” ACS Chemical Bbiology, vol. 2, no. 4, pp. 231-236, April, 2007.
[32] E. Braungart, M. Gerlach, P. Riederer, R. Baumeister, and M. C. Hoener, “Caenorhabditis elegans MPP+ model of Parkinson’s disease for high-throughput drug screenings,” Neurodegenerative Diseases, vol. 1, no. 4-5, pp. 175-183, November, 2004.
[33] E. M. Jorgensen, and S. E. Mango, “The art and design of genetic screens: Caenorhabditis elegans,” Nature Reviews Genetics, vol. 3, no. 5, pp. 356-369, May, 2002.
[34] J. C. Gilbert, J. Hadgraft, A. Bye, and L. G. Brookes, “Drug release from Pluronic F-127 gels,” International Journal of Pharmaceutics, vol. 32, no. 2, pp. 223-228, October, 1986.
[35] H. Geng, H. Song, J. Qi, and D. Cui, “Sustained release of VEGF from PLGA nanoparticles embedded thermo-sensitive hydrogel in full-thickness porcine bladder acellular matrix,” Nanoscale Research letters, vol. 6, no. 1, pp. 1-8, December, 2011.
[36] S. T. Henderson, and T. E. Johnson, “daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans,” Current Biology, vol. 11, no. 24, pp. 1975-1980, December, 2001.
[37] L. A. Herndon, P. J. Schmeissner, J. M. Dudaronek, P. A. Brown, K. M. Listner, Y. Sakano, M. C. Paupard, D. H. Hall, and M. Driscoll, “Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans,” Nature, vol. 419, no. 6909, pp. 808-814, October, 2002.
[38] A. Ben-Zvi, E. A. Miller, and R. I. Morimoto, “Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging,” Proceedings of the National Academy of Sciences, vol. 106, no. 35, pp. 14914-14919, September 2009.
[39] M. Porta-de-la-Riva, L. Fontrodona, A. Villanueva, and J. Cerón, “Basic Caenorhabditis elegans methods: synchronization and observation,” JoVE (Journal of Visualized Experiments), no. 64, pp. e4019-e4019, July, 2012.
[40] M. D. Abràmoff, P. J. Magalhães, and S. J. Ram, “Image processing with ImageJ,” Biophotonics International, vol. 11, no. 7, pp. 36-42, July, 2004.
[41] J. T. Pierce-Shimomura, B. L. Chen, J. J. Mun, R. Ho, R. Sarkis, and S. L. McIntire, “Genetic analysis of crawling and swimming locomotory patterns in C. elegans,” Proceedings of the National Academy of Sciences, vol. 105, no. 52, pp. 20982-20987, December, 2008.
[42] H.-S. Chuang, D. M. Raizen, A. Lamb, N. Dabbish, and H. H. Bau, “Dielectrophoresis of Caenorhabditis elegans,” Lab on a Chip, vol. 11, no. 4, pp. 599-604, January, 2011.
[43] D. Ross, M. Gaitan, and L. E. Locascio, “Temperature measurement in microfluidic systems using a temperature-dependent fluorescent dye,” Analytical Chemistry, vol. 73, no. 17, pp. 4117-4123, July, 2001.
[44] R. F. Kubin, and A. N. Fletcher, “Fluorescence quantum yields of some rhodamine dyes,” Journal of Luminescence, vol. 27, no. 4, pp. 455-462, December, 1983.
[45] S. L. Edwards, N. K. Charlie, M. C. Milfort, B. S. Brown, C. N. Gravlin, J. E. Knecht, and K. G. Miller, “A novel molecular solution for ultraviolet light detection in Caenorhabditis elegans,” PLoS Biol, vol. 6, no. 8, pp. e198, August, 2008.
[46] A. Ward, J. Liu, Z. Feng, and X. S. Xu, “Light-sensitive neurons and channels mediate phototaxis in C. elegans,” Nature Neuroscience, vol. 11, no. 8, pp. 916-922, July, 2008.
[47] R. Ghosh, and S. W. Emmons, “Episodic swimming behavior in the nematode C. elegans,” Journal of Experimental Biology, vol. 211, no. 23, pp. 3703-3711, October 2008.
[48] J. Korta, D. A. Clark, C. V. Gabel, L. Mahadevan, and A. D. Samuel, “Mechanosensation and mechanical load modulate the locomotory gait of swimming C. elegans,” Journal of Experimental Biology, vol. 210, no. 13, pp. 2383-2389, April, 2007.
[49] S. L. Mclntire, E. Jorgensen, J. Kaplan, and H. R. Horvitz, “The GABAergic nervous system of Caenorhabditis elegans,” Nature, vol. 364, pp. 337-341, July, 1993.
[50] N. Croll, “Components and patterns in the behaviour of the nematode Caenorhabditis elegans,” Journal of Zoology, vol. 176, no. 2, pp. 159-176, June, 1975.
[51] W.-J. Kuo, Y.-S. Sie, and H.-S. Chuang, “Characterizations of kinetic power and propulsion of the nematode Caenorhabditis elegans based on a micro-particle image velocimetry system,” Biomicrofluidics, vol. 8, no. 2, pp. 024116, April, 2014.
[52] Y.-S. Sie, and H.-S. Chuang, “A micro-volume viscosity measurement technique based on μPIV diffusometry,” Microfluidics and Nanofluidics, vol. 16, no. 1-2, pp. 65-72, January, 2014.
[53] R. D. Keane, and R. J. Adrian, “Theory of cross-correlation analysis of PIV images,” Applied Scientific Research, vol. 49, no. 3, pp. 191-215, July, 1992.
[54] E. Kim, L. Sun, C. V. Gabel, and C. Fang-Yen, “Long-term imaging of Caenorhabditis elegans using nanoparticle-mediated immobilization,” PloS One, vol. 8, no. 1, pp. e53419, January, 2013.
[55] K. Nowak, K. McCullagh, E. Poon, and K. E. Davies, "Muscular dystrophies related to the cytoskeleton/nuclear envelope." Novartis Foundation Symposium, vol. 264, p. 98-117,2006.
[56] U. T. Brunk, and A. Terman, “Lipofuscin: mechanisms of age-related accumulation and influence on cell function,” Free Radical Biology and Medicine, vol. 33, no. 5, pp. 611-619, September, 2002.
[57] G. V. Clokey, and L. A. Jacobson, “The autofluorescent “lipofuscin granules” in the intestinal cells of Caenorhabditis elegans are secondary lysosomes,” Mechanisms of Ageing and Development, vol. 35, no. 1, pp. 79-94, June, 1986.
[58] T. Murayama, and I. N. Maruyama, “Decision making in C. elegans chemotaxis to alkaline pH: competition between two sensory neurons, ASEL and ASH,” Communicative & Integrative Biology, vol. 6, no. 6, pp. 1007-12, september, 2013.
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