臺灣博碩士論文加值系統

血癌又稱為白血病，是指人體骨髓造血幹細胞（Hematopoietic stem cells,簡稱HSC）DNA變異產生癌化病變，不停地製造不正常之白血球，使得正常的白血球數量減少，不正常的白血球不斷的增生，於是將整個骨髓佔據並偏佈於血液中，已經嚴重影響正常血球的功能，導致身體很容易受到細菌感染。由於自然殺手細胞治療法副作用小，治療期間可維持病患相對較良好的生活品質，因此近年來有越來越多的患者選擇使用自然殺手細胞療法治療血癌。
本研究利用微機電製程技術製作一微流體細胞晶片，藉由微流道設計與微流體操控技術，達到捕捉懸浮性細胞的功能。於細胞晶片中持續供應含有介白素配方之細胞培養液活化人類自然殺手細胞，並量測與分析細胞激素提升人類自然殺手細胞株(NK92-MI)對血癌細胞株(K562)之毒殺能力。以不同效應細胞對目標細胞比例(Effector to target cell ratio, E/T ratio) 混和及不同反應時間培養之樣品量測毒殺能力並分析其結果，利用流式細胞儀提供定量化資料，並與細胞晶片分析細胞激素活化之人類自然殺手細胞株(NK-92MI)對血癌細胞株(K562)毒殺能力之結果差異性作比較，以驗證細胞晶片分析毒殺能力之可行性。此外，藉由整合倒立式高解析度共軛焦顯微鏡，利用共軛焦顯微鏡掃描樣本不同的共軛焦面再由電腦重建影像的方式與螢光染色技術，可得到極高解析度與對比度之螢光影像，可做長期的追蹤觀測細胞間之反應行為，並將反應過程以影像紀錄，為自然殺手細胞毒殺癌細胞毒殺過程之定量化資訊。
由結果顯示，K562被毒殺隨著E/T ratio的增加有提高之趨勢，亦即自然殺手細胞越多，則血癌細胞被毒殺的比例愈高。此外，於E/T ratio為2:1時，使用細胞晶片量測毒殺能力之結果與流式細胞儀所量測之結果最為相近。在分別將各組樣品以不同時間培養後量測毒殺能力，隨著2小時至18小時之量測結果，毒殺比率隨時間增加有提高趨勢，但趨勢漸緩，亦即毒殺能力有其限制。而於第18小時所量測毒殺率之結果顯示，加入介白素IL-12所激活之自然殺手細胞毒殺率相較於未使用者，其毒殺率從49.7%提升至79.2%，顯示藉由細胞激素IL-12激活自然殺手細胞效果顯著。將毒殺反應過程由共軛焦顯微鏡下觀察，K562細胞被NK92-MI細胞毒殺時，細胞形態上逐漸變化，包括:細胞萎縮、細胞膜出芽表現、及由凋亡細胞所產生的細胞凋亡體(apoptosis body)，其長期追蹤觀測細胞間反應行為之定性資訊可佐證定量之結果。

Leukemia, known as blood cancer, occurs due to DNA variation of human bone marrow stem cells, making too many immature white blood cells. A large number of these immature white blood cells present in the blood seriously affect the normal function of blood cells. Due to little side effect of natural killer cell, natural killer cell therapy has been chosen as an auxiliary strategy in post chemical treatment for good quality of life.
In this study, a microfluidic cell-trapped device was designed and manufactured by MEMS technique. With the design of narrow gaps between channels, suspension cells were trapped by a fluidic pressure drop between channels to be capable of trapped cell monitoring or observation under a microscopy. As a result, cell-cell interaction and cell apoptosis processes can be investigated with this device under a currently used fluorescence microscopy. In this study, cytokine interleukin 12 (IL 12) has been chosen to activate human natural killer cells in killing cancer cell (K562) process. With various ratios of effector cells to target cells (E/T ratio) in a cell-trapped device, a microfluidic cytotoxic assay was analyzed under a fluorescence microscopy. The cytotoxic results of the present device showed an excellent agreement with those by the conventional flow cytometry. The merit of the present device shows a feasible cytotoxic analysis using few cells in an order of 102 instead of around 106 cells for analysis in conventional flow cytometry. Additionally, the cytotoxicity of NK cells against cancer cells (K562) was found in a range from 49.7% to 79.2% over time periods in a cytokine IL-12 cell activation. Moreover, trapped in a device with an inverted high-resolution confocal microscopy by scanning fluorescent staining cell samples, the on-chip NK and cancer mixed cells in a cytokine-activated medium fluidics were kept monitoring in a long-term tracking of cell killing activities. Cell morphology was found to gradually change over time. Cell apoptosis process of cell shrinkage, membrane budding, and apoptosis bodies were firstly found with the present device in this experiment.
Lastly, the on-chip determination of cell killing process and activity has been firstly achieved by this present device.

誌謝 III
中文摘要 V
ABSTRACT VII
目錄 V
圖目錄 IX
第1章 緒論 1
1.1前言 1
1.2 研究動機 6
1.3 文獻回顧 9
1.3.1 自然殺手細胞特性 9
1.3.2 自然殺手細胞植入療法 11
1.3.3 生物晶片細胞操控技術 15
1.3.3 共軛焦顯微鏡應用於自然殺手細胞毒殺血癌細胞觀測 19
1.4 論文架構 22
第2章 研究原理 23
2.1 微流道流場理論分析 23
2.1.1 水力聚焦流場分析 23
2.1.2 流道捕捉細胞壓力差與細胞受力分析 26
2.2 細胞株介紹 29
2.2.1 自然殺手細胞株(NK-92MI) 29
2.2.2 血癌細胞株(K562) 30
2.3 免疫細胞化學染色技術 31
2.3 細胞骨架介紹與螢光染色 33
2.3.1 細胞骨架介紹 33
2.3.2 細胞骨骼螢光染色 35
2.5 細胞凋亡與標定 36
2.4 血球計數盤 38
2.7流式細胞技術 40
2.7.1液流系統(FLUIDICS) 41
2.7.2光學及電子系統(OPTICS AND ELECTRONICS) 41
2.8 共軛焦顯微鏡 42
2.8.1 共軛焦顯微鏡基本原理 42
2.8.2 ZEISS LSM780共軛焦顯微鏡介紹 44
第3章 研究方法 45
3.1 實驗架構 45
3.2 微流道細胞晶片設計與製程 47
3.2.1 細胞晶片設計 47
3.2.2 細胞晶片微流道母模製程 49
3.2.3 細胞晶片製作封裝 52
3.3 細胞培養 55
3.3.1細胞株解凍與培養 55
3.3.2 NK-92MI自然殺手細胞株培養 56
3.3.3 K562血癌細胞株培養 58
3.4 細胞樣品製備與實驗操作流程 60
3.4.1 細胞螢光標定 60
3.4.2 流式細胞儀之細胞毒殺率分析 63
3.5 共軛焦顯微鏡即時影像系統架設 64
第4章 結果與討論 67
4.1 不同效應對目標細胞比例下之細胞毒殺率分析 67
4.2 IL-12活化NK-92MI細胞對K562細胞毒殺能力分析 69
4.3 共軛焦顯微鏡毒觀察細胞毒殺過程與分析 71
4.3.1螢光染色測試 72
4.3.2 180~250分鐘細胞毒殺反應追蹤觀察 73
4.2.3 180~320分鐘細胞毒殺反應追蹤觀察 76
4.3.4 330~435分鐘細胞毒殺反應追蹤觀察 79
第5章 結論與未來展望 81
5.1 結論 81
5.2 未來展望 83
5.2.1生物晶片與系統改善 83
5.2.2未來應用 83
附錄 85
附錄1 85
附錄2 86
參考文獻 89

[1]Available: http://www.chin-i.com.tw/epaper_detail.php?id=474&;cid=2
[2]D.A. Pollyea, H. E. Kohrt, and B. C. Medeiros, "Acute myeloid leukemia in the elderly: a review," Br J Haematol, vol. 152, pp. 524-42, 2011.
[3]C. Fausel, "Targeted chronic myeloid leukemia therapy: seeking a cure," American Journal of Health-System Pharmacy, vol. 64, pp. S9-S15, 2007.
[4]International Agency for Research on Cancer. Available: http://www-dep.iarc.fr/
[5]A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, and M. J. Thun, "Cancer statistics, 2009," CA Cancer J Clin, vol. 59, pp. 225-49, 2009.
[6]行政院衛生署中華民國99年癌症登記報告。
[7]National Institutes of Health. Available: http://www.nih.gov/
[8]T. Kawase, Y. Morishima, K. Matsuo, K. Kashiwase, H. Inoko, H. Saji, S. Kato,T. Juji, Y. Kodera, T. Sasazuki, "High-risk HLA allele mismatch combinations responsible for severe acute graft-versus-host disease and implication for its molecular mechanism," Blood, vol. 110, no. 7, pp. 2235–2241, 2007.
[9]L. Ruggeri, A. Mancusi, K. Perruccio, E. Burchielli, M. F. Martelli, and A. Velardi, "Natural killer cell alloreactivity for leukemia therapy," J Immunother, vol. 28, pp. 175-82, 2005.
[10]J.E. Rubnitz, H. Inaba, R. C. Ribeiro, S. Pounds, B. Rooney, T. Bell, C.H. Pui, and W. Leung, "NKAML: A pilot study to determine the safety and feasibility of haploidentical natural killer cell transplantation in childhood acute myeloid leukemia," J Clin Oncol, vol. 28, pp. 955-959, 2010.
[11]G. Suck, "Novel approaches using natural killer cells in cancer therapy," Seminars in Cancer Biology, vol. 16, pp. 412-418, 2006.
[12]L. Ruggeri, A. Mancusi, E. Burchielli, F. Aversa, M. F. Martelli, and A. Velardi, “Natural killer cell alloreactivity in allogeneic hematopoietic transplantation,” Current Opinion in Oncology, vol. 19, no. 2, pp. 142–147, 2007.
[13]A. McGrady, P. Conran, D. Dickey, D. Garman, E. Farris,
C.S. Brzezinski, "The effects of biofeedback-assisted relaxation on cell-mediated immunity, cortisol, and white blood cell count in healthy adult subjects," J Behav Med, vol. 15(4), pp.343-54,1992.
[14]L. Ruggeri, A. Mancusi, M. Capanni, E. Urbani, A. Carotti, T. Aloisi, M. Stern, D. Pende, K. Perruccio, E. Burchielli, F. Topini, E. Bianchi, F. Aversa, M.F. Martelli, A. Velardi, ''Donor Natural Killer Cell Allorecognition of Missing Self in Haploidentical Hematopoietic Transplantation for Acute Myeloid Leukemia: Challenging Its Predictive Value,'' Blood, vol. 110, pp. 433-40, 2007.
[15]K. Guldevall, B. Vanherberghen, T. Frisk, J. Hurtig, A. Christakou, O. Manneberg, S. Lindstrom, H. Andersson Svahn, M. Wiklund, B. Onfelt, "imaging immune surveillance of individual natural killer cells confined in microwell arrays," PLoS ONE, nol. 5, p. e15453, 2010.
[16]S.F. Sherif, A.C. Michael, "Human natural killer cell development and biology," Blood reviews, vol. 20, pp. 123-137, 2006.
[17]M.A. Cooper, T.A. Fehniger, M.A. Caligiuri, "The biology of human natural killer-cell subsets," Trends Immunol, vol. 22, pp.633–640, 2001.
[18]A. Chan, D.L. Hong, A. Atzberger, S. Kollnberger, A.D. Filer, C.D. Buckley,
A. McMichael, T. Enver, P. Bowness "CD56bright human NK cells differentiate into CD56dim cells: role of contact with peripheral fibroblasts," J Immunol, vol. 179, pp. 89-94, 2007.
[19]J.E. Rubnitz, H. Inaba, R. C. Ribeiro, S. Pounds, B. Rooney, T. Bell, C.-H. Pui, and W. Leung, "NKAML: A Pilot Study to Determine the Safety and Feasibility of Haploidentical Natural Killer Cell Transplantation in Childhood Acute Myeloid Leukemia," J Clin Oncol, vol. 28, pp. 955-959, 2010.
[20]G. Suck, "Novel approaches using natural killer cells in cancer therapy," Seminars in Cancer Biology, vol. 16, pp. 412-418, 2006.
[21]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.
[22]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.
[23]R. Bhat, C. Watzl, "Serial killing of tumor cells by human natural killer cells--enhancement by therapeutic antibodies," PLoS One, vol. 2, p. e326, 2007.
[24]R. Castriconi, A. Dondero, M.V. Corrias, E. Lanino, D. Pende, L. Moretta,
C. Bottino, and A. Moretta, "Natural killer cell-mediated killing of freshly isolated neuroblastoma cells," Cancer Research, vol. 64, p. 9180, 2004.
[25]J.H. Gong, G. Maki, H.G. Klingemann, "Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells," Leukemia, vol. 8(4), pp.652-658, 1994.
[26]M. Yamada, M. Seki, "Hydrodynamic filtration for on-chip particle concentration and classification utilizing microfluidics," Lab on a Chip, vol. 5, pp. 1233-1239, 2005.
[27]M. Yang, C.W. Li, 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.
[28]D.D. Carlo, L.Y. Wu, L.P. Lee, "Dynamic single cell culture array," Lab on a Chip, vol. 6, pp. 1445-1449, 2006.
[29]P.P. Banerjee, R. Pandey, R. Zheng, M.M. Suhoski, L. Monaco-Shawver, J.S. Orange, "Cdc42-interacting protein – 4 functionally links actin and microtubule networks at the cytolytic NK cell immunological synapse," JEM, Vol. 204, pp. 2305-2320, 2007.
[30]K. RadoSevic, M.T. van Leeuwen, I.M. Seers-Nolten, C.G. Figdor, B.G. de Grooth, J. Greve, "Changes in actin organization during the cytotoxic process," Cytometry , vol. 15, pp. 320-326, 1994.
[31]R.B. da Silva, C. Graf, C. Mu &;#776;nz1, "Cytoskeletal stabilization of inhibitory interactions in immunologic synapses of mature human dendritic cells with natural killer cells," blood, vol. 118, pp. 6487-6498, 2011.
[32]F.L. Lai, "A study on cell-based biosensor for cytotoxic assay of human nature killer cells against cancer cells," Doctoral dissertation, NTU, 2011.
[33]C.T. Lim, M. Dao, S. Suresh, C.H. Sow, K.T. Chew, "Large deformation of living cells using laser traps," Acta Materialia , vol 52, pp.1837–1845, 2004.
[34]C.B. Lozzio, B.B. Lozzio, "Human chronic myelogenous leukemia cell-line with positive philadelphia chromosome," Blood, vol. 45, pp. 321-34, 1975.
[35]J.H. Gong, G. Maki, H.G. Klingemann, "Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells," Leukemia, vol. 8(4), pp.652-658, 1994.
[36]Y.K. Tam, G. Maki, B. Miyagawa, B. Hennemann, T. Tonn, H.G. Klingemann "Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy," Hum. Gene Ther, vol.10, pp. 1359-1373, 1999.
[37]H.P. Koeffler, D.W. Golde, "Human myeloid leukemia cell lines: a review," Blood, vol.56, pp. 344-350, 1980.
[38]L.C. Javois,"Immunocytochemistry Methods and Protocols," 2nd edition, 1999.
[39]J.E. Coligan, A.M. Kbuisbeek, D.H. Marculies, E.M. Shevach, W. Strober "Current Protocols in Immunology," Greene Publishing Associates and Wiley-Interscience, NY, 1991.
[40]Available: http://www.biotechland.it/index.html
[41]Life Technologies’s web, Available: http://www.lifetechnologies.com/order/catalog/product/T34075
[42]A. Gewies, "Introduction to apoptosis," Apo Review, Cell death, 2003.
[43]G. Majno and I. Joris, "Apoptosis, oncosis, and necrosis. An overview of cell death," Am J Pathol, vol. 146, pp. 3-15, Jan 1995.
[44]A. Gewies, "Introduction to apoptosis," Apo Review, 2003.
[45]M. Brown, C. Wittwer, "Flow cytometry: principles and clinical applications in hematology," Clin Chem, vol. 46, pp. 1221-9, 2000.
[46]J.B. Pawley,"Handbook of Biological Confocal Microscopy (3rd ed.),"Berlin: Springer, 2006.
[47]M. Minsky, Ameter, Levy, US Patent 3.013.467, 1957.
[48]ZEISS LSM780 Product information, Available: http://corporate.zeiss.com
[49]O. Holub, "Fluorescence polarization/anisotropy imaging with LSM 710 / LSM 780," ZEISS White Paper, 2012.
[50]C.K. Cheng, "A Real-Time Investigation on Cytotoxic Assay of Human Natural Killer Cells against Cancer Cells in Microfluidic Device by Inverted Point Laser Scanning Confocal Microscopy", Master’s thesis, NTU, 2013.
[51]Y.T. Liu, "A Study on Cytotoxic Analysis of Cytokine-activated Human Natural Killer Cells by Cell-based Microdevice", Master’s thesis, NTU, 2012.