請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19161
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃榮山(Long-Son Huang) | |
dc.contributor.author | Yi-Ling Li | en |
dc.contributor.author | 李宜霖 | zh_TW |
dc.date.accessioned | 2021-06-08T01:47:07Z | - |
dc.date.copyright | 2016-10-14 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-08 | |
dc.identifier.citation | [1] 臺北市立聯合醫院癌症防治研究發展中心. Available: http://website.tpech.gov.tw/cancer/.
[2] International Agency for Research on Cancer. Available: http://www.iarc.fr/. [3] 行政院衛生署中華民國102年癌症登記報告. [4] T. Kawase, Y. Morishima, K. Matsuo, K. Kashiwase, H. Inoko, H. Saji, et al., 'High-risk HLA allele mismatch combinations responsible for severe acute graft-versus-host disease and implication for its molecular mechanism,' Blood, vol. 110, pp. 2235-2241, 2007. [5] L. Ruggeri, A. Mancusi, K. Perruccio, E. Burchielli, M. F. Martelli, and A. Velardi, 'Natural killer cell alloreactivity for leukemia therapy,' Journal of Immunotherapy, vol. 28, pp. 175-182, 2005. [6] J. E. Rubnitz, H. Inaba, R. C. Ribeiro, S. Pounds, B. Rooney, T. Bell, et al., 'NKAML: a pilot study to determine the safety and feasibility of haploidentical natural killer cell transplantation in childhood acute myeloid leukemia,' Journal of Clinical Oncology, vol. 28, pp. 955-959, 2010. [7] G. Suck, 'Novel approaches using natural killer cells in cancer therapy,' in Seminars in cancer biology, 2006, pp. 412-418. [8] A. McGrady, P. Conran, D. Dickey, D. Garman, E. Farris, and C. Schumann-Brzezinski, 'The effects of biofeedback-assisted relaxation on cell-mediated immunity, cortisol, and white blood cell count in healthy adult subjects,' Journal of Behavioral Medicine, vol. 15, pp. 343-354, 1992. [9] S. S. Farag and M. A. Caligiuri, 'Human natural killer cell development and biology,' Blood reviews, vol. 20, pp. 123-137, 2006. [10] I.-T. Kao, C.-L. Yao, Z.-L. Kong, M.-L. Wu, T.-L. Chuang, and S.-M. Hwang, 'Generation of natural killer cells from serum-free, expanded human umbilical cord blood CD34+ cells,' Stem cells and development, vol. 16, pp. 1043-1052, 2007. [11] R. Bhat and C. Watzl, 'Serial killing of tumor cells by human natural killer cells–enhancement by therapeutic antibodies,' PLoS One, vol. 2, p. e326, 2007. [12] K. Takahashi, A. Hattori, I. Suzuki, T. Ichiki, and K. Yasuda, 'Non-destructive on-chip cell sorting system with real-time microscopic image processing,' Journal of nanobiotechnology, vol. 2, p. 1, 2004. [13] M. Yamada and M. Seki, 'Hydrodynamic filtration for on-chip particle concentration and classification utilizing microfluidics,' Lab on a Chip, vol. 5, pp. 1233-1239, 2005. [14] M. Yang, C.-W. Li, and J. Yang, 'Cell docking and on-chip monitoring of cellular reactions with a controlled concentration gradient on a microfluidic device,' Analytical chemistry, vol. 74, pp. 3991-4001, 2002. [15] International Telecommunication Union. Available: https://www.itu.int/en/ITU-D/Statistics/Documents/facts/ICTFactsFigures2015.pdf. [16] D. N. Breslauer, R. N. Maamari, N. A. Switz, W. A. Lam, and D. A. Fletcher, 'Mobile phone based clinical microscopy for global health applications,' PloS one, vol. 4, p. e6320, 2009. [17] H. C. Koydemir, Z. Gorocs, D. Tseng, B. Cortazar, S. Feng, R. Y. L. Chan, et al., 'Rapid imaging, detection and quantification of Giardia lamblia cysts using mobile-phone based fluorescent microscopy and machine learning,' Lab on a chip, vol. 15, pp. 1284-1293, 2015. [18] H. Zhu, O. Yaglidere, T.-W. Su, D. Tseng, and A. Ozcan, 'Cost-effective and compact wide-field fluorescent imaging on a cell-phone,' Lab on a Chip, vol. 11, pp. 315-322, 2011. [19] 科技部高瞻自然科學教學平台. Available: http://highscope.ch.ntu.edu.tw/ [20] J. J. Wang, 'A real-time Investigation on Cytotoxic Assay of Primary Human Natural Killer Cells against Leukemic Cells in Microfluidic Device,' master dissertation, 2015. [21] F. L. Lai, 'A study on cell-based biosensor for cytotoxic assay of human nature killer cells against cancer cells,' Doctoral dissertation, vol. NTU, 2011. [22] C. Lim, M. Dao, S. Suresh, C. Sow, and K. Chew, 'Large deformation of living cells using laser traps,' Acta Materialia, vol. 52, pp. 1837-1845, 2004. [23] J.-H. Gong, G. Maki, and H. Klingemann, 'Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells,' Leukemia, vol. 8, pp. 652-658, 1994. [24] Y. Tam, G. Maki, B. Miyagawa, B. Hennemann, T. Tonn, and H.-G. Klingemann, 'Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy,' Human gene therapy, vol. 10, pp. 1359-1373, 1999. [25] C. B. Lozzio and B. B. Lozzio, 'Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome,' Blood, vol. 45, pp. 321-334, 1975. [26] J. J. G. Glover, F. Netter, 'Citoscheletro,' BiotechLand, Available: http://www.biotechland.it/index.html. [27] A. F. Pegoraro, A. Ridsdale, D. J. Moffatt, J. P. Pezacki, and A. Stolow, 'High performance multimodal CARS microscopy using a single femtosecond source,' in SPIE BiOS: Biomedical Optics, 2009, pp. 71830Z-71830Z-9. [28] G. Majno and I. Joris, 'Apoptosis, oncosis, and necrosis. An overview of cell death,' The American journal of pathology, vol. 146, p. 3, 1995. [29] A. Lawen, 'Apoptosis—an introduction,' Bioessays, vol. 25, pp. 888-896, 2003. [30] M. Brown and C. Wittwer, 'Flow cytometry: principles and clinical applications in hematology,' Clinical chemistry, vol. 46, pp. 1221-1229, 2000. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19161 | - |
dc.description.abstract | 近年來有越來越多的患者在免疫細胞治療中,選擇以自身免疫系統的自然殺手細胞體外增生,再注入體內毒殺血癌細胞治療白血病,免疫自然殺手細胞療法副作用小,在治療期間對病患來說相對其他傳統的化學療法有較良好的生活品質。在免疫細胞治療過程中,體外增生的免疫自然殺手細胞,注入體內前常需檢測其增生細胞之毒殺能力與活性,傳統上使用流式細胞儀分析細胞毒殺能力,需要大量的細胞數量(105~106)方能做可靠性的檢測,但對病患來說,增生後臨床用的自然殺手細胞數量有限、非常珍貴且昂貴,因此不適合挪用臨床用約百萬顆大量細胞作毒殺檢測之用。
本研究開發創新的光學系統裝置與細胞晶片,可作為僅需少量細胞(約102顆)之細胞毒殺檢測、即時觀測系統與即時雲端影像傳輸之智慧型手機螢光顯微系統裝置。本研究係利用(1)智慧型手機本身可做數位影像拍攝、自動對焦及雲端傳輸功能,(2)創新設計光學系統,類似倒立式螢光顯微光學之微型模組,包含LED光源、濾片及鏡片模組等裝置,(3)再利用微機電技術製作無幫浦微流體生醫晶片,藉由水柱高壓力驅動流體,配合微流道結構設計與微流體操控技術捕捉細胞,(4)以免疫細胞治療之自然殺手細胞與血癌細胞之毒殺作用並分析。整合以上四大項,以智慧型手機為基礎裝置,建構一個即時細胞檢測、螢光觀測與即時雲端影像傳輸之可攜式微型系統。 在微流體晶片,以二維矩形微流道中的流體水力動力聚焦理論模型,透過晶片中的管柱高度決定壓力與流速,產生水力聚焦現象,並設計微流道之間與間隙,透過流體壓力差,細胞會被吸引往間隙前進,由於細胞直徑(~10μm)大於縫隙高度(4μm),因此細胞會被捕捉於主流道側壁的反應區,細胞間進行毒殺作用。 再者,創新之光學路徑設計、材料選擇,測試鏡頭的光學品質並計算其調制轉換函數約為0.2,最後透過光學元件的組裝並與無幫浦微流體生物晶片整合,結合成智慧型手機螢光顯微系統裝置與細胞晶片檢測,首先以螢光微珠作測試,測量此系統觀測視野區之光學特性並與傳統螢光顯微鏡影像比較,再進一步地測試真實的螢光染色細胞,可成功地測試螢光細胞流入微流體晶片。最後,以此創新之智慧型手機螢光顯微系統裝置與細胞晶片,利用自然殺手細胞及血癌細胞在此微型系統進行毒殺作用,以效能細胞(自然殺手細胞)對目標細胞(血癌細胞)比例為1:1的條件,在不同毒殺時間下,結果發現毒殺時間越長,其毒殺率也越高,並且將相同的細胞樣本以流式細胞儀進行分析比較,實驗結果具一致性。 本研究成功地以創新智慧型手機螢光顯微光學模組,取代傳統的螢光顯微鏡和微流體生物晶片,其功能並可取代傳統流式細胞儀之螢光細胞毒殺分析,此創新之細胞檢測系統,成功地運用少量細胞(~102)分析出細胞毒殺率,解決流式細胞儀需要大量細胞(~106)檢測的缺點,極適合臨床應用,其微型化攜帶方便且能即時遠端檢測,可應用於食品販賣現場檢測、實地環境檢測或科學教育之應用。 | zh_TW |
dc.description.abstract | In recent years, a growing number of patients were cured by nature killer cell transplantation. Due to the little side effect of natural killer cells in cell transplantation, natural killer cell therapy has been chosen as an auxiliary strategy in post chemical treatment for good quality of life. Traditionally, cytotoxicity analysis is performed by using flow cytometry; however, it requires a large number of cells (~106). In clinical fields, proliferated nature killer cells are still limited and expensive, which remains a challenge for cytotoxic investigation by acquiring part of proliferated NK cells.
In this study, the new smartphone-based fluorescence microscopy integrated with a microfluidic cell device was designed to be optically inverted by assembling highly bright LED, miniaturized lens module, and filter. Meanwhile, a microfluidic cell-trapped device was designed and manufactured by MEMS technique. With the design of microchannels, suspension nature killer cells and cancer cells were trapped by a fluidic pressure drop in microchannels. The miniature smartphone-based, inverted fluorescent microscopic system exhibited a real-time monitoring, cloud image computing, and cytotoxic analysis. In optics, optical quality and performance of the inverted smartphone-based fluorescence microscopy was characterized by area of interest, enlargement, and image resolution. The quality of lens was investigated by modulation transfer function (MTF) to be around 0.2 in MTF. In fluorescence characterization, fluorescent microbeads were used to characterize the area of interest in this microscopic system. The area of interest was found to be 2.5 mm2. At last, the cytotoxic analysis was performed by introducing nature killer cells and cancer cells into the microfluidic cell device. With the effector-to-target cell ratio of 1 between both cell groups, the cytotoxic result of the present system showed an excellent agreement with that in flow cytometer. This study successfully demonstrated the cell monitoring and investigation of the inverted smartphone-based fluorescence microscopy with the microfluidic cell device. In addition, the miniature integrated system presented the excellence in cytotoxic analysis in a small amount of cells (~102) in comparison with that in flow cytometry. With the integration of the smartphone and optical module, this new system can be wirelessly transmitted to the cloud for image computing and for remote detection. The present system creates a new platform of a portable, cost-effective inverted fluorescence microscopy, cell investigation, real-time field test with cloud image computing, and medical examination. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:47:07Z (GMT). No. of bitstreams: 1 ntu-105-R03543008-1.pdf: 3984661 bytes, checksum: f2fbae1d84ee079370ef01f5c64b129f (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 目錄
誌謝 i 中文摘要 ii Abstract iv 目錄 vi 圖目錄 viii 表目錄 xi 第1章 緒論 1 1-1 前言 1 1-2 研究動機 4 1-3 文獻回顧 6 1-3.1 自然殺手細胞特性 6 1-3.2 自然殺手細胞療法 7 1-3.3 生物晶片細胞操控技術 9 1-3.4 手機螢光顯微鏡應用 12 1-4 論文架構 15 第2章 原理 16 2-1 螢光顯微鏡 16 2-1.1 螢光顯微鏡基本原理 16 2-1.2 手機螢光顯微鏡 18 2-2 微流道流場理論分析 19 2-2.1 水力聚焦流場分析 19 2-2.2 流道捕捉細胞壓力差與細胞受力分析 21 2-3 細胞株介紹 24 2-3.1 自然殺手細胞(NK-92MI) 24 2-3.2 血癌細胞株(K562) 24 2-4 細胞骨骼介紹與螢光標定 25 2-4.1 細胞骨骼介紹 25 2-4.2 細胞骨骼螢光染色 27 2-5 細胞凋亡機制與標定 28 2-5.1 細胞凋亡機制 28 2-5.2 細胞凋亡之螢光標定 29 2-6 流式細胞分析儀 30 第3章 研究方法 32 3-1 實驗架構 32 3-2 手機螢光顯微鏡設計與架設 33 3-2.1 手機螢光顯微鏡設計 33 3-2.2 手機螢光顯微鏡架設 36 3-3 微流道生物晶片設計與製程 39 3-3.1 微流道生物晶片設計 39 3-3.2 微流道生物晶片母模製程 40 3-3.3 微流道生物晶片製作封裝 43 3-4 細胞培養 46 3-5 細胞樣品製備 48 3-5.1 血癌細胞株K562細胞骨骼螢光標定 48 3-5.2 細胞凋亡螢光標定 49 3-6 實驗操作流程 50 3-6.1 手機螢光顯微鏡可行性 50 3-6.2 微流體生物晶片毒殺分析 51 3-6.3 流式細胞儀毒殺分析 52 第4章 結果與討論 53 4-1 細胞捕捉機制與受力分析 53 4-1.1 細胞捕捉機制 53 4-1.2 細胞受力分析 53 4-2 鏡頭解析度測試 56 4-3 手機螢光顯微鏡測試 58 4-3.1 螢光微珠測試 58 4-3.2 影像處理與分析 60 4-3.3 染色細胞測試 61 4-3.4 微流道生物晶片捕捉觀察 62 4-4 流式細胞儀之毒殺率計算 63 4-5 生物晶片配合手機顯微鏡之毒殺計算 65 第5章 結論與未來展望 69 5-1 結論 69 5-2 未來展望 71 References 72 | |
dc.language.iso | zh-TW | |
dc.title | 手機螢光顯微鏡及雲端影像傳輸應用於微流體晶片中細胞毒殺之系統開發與研究 | zh_TW |
dc.title | A Smartphone-based Fluorescent Microscopy with Cloud Image Processing for Pump-free Microfluidic Cytotoxic Assays | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃君偉(Jiun-Woei Huang),蔡博宇(Bor-Yu Tsai) | |
dc.subject.keyword | 自然殺手細胞,手機螢光顯微鏡,生物晶片,微機電製程,即時觀測系統, | zh_TW |
dc.subject.keyword | Natural killer cells,phone-based fluorescent microscope,microfluidic device,MEMS,real-time observation system, | en |
dc.relation.page | 75 | |
dc.identifier.doi | 10.6342/NTU201601349 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-08-08 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 應用力學研究所 | zh_TW |
顯示於系所單位: | 應用力學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-105-1.pdf 目前未授權公開取用 | 3.89 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。