簡易檢索 / 詳目顯示

研究生: 涂夏爾
Punde Tushar Harishchandra
論文名稱: 微流體生醫晶片應用於誘發肺部發炎之趨化蛋白質研究
Microfluidics-based Dynamic Migration Labchip for Studying Protein-induced Lung Inflammation
指導教授: 劉承賢
Liu, Cheng-Hsien
口試委員: 凌永健
Ling, Yong-Chien
莊校奇
Chuang, Hsiao-Chi
王圳華
Wang, Chun-Hua
陳致真
Chen, Chih-Chen
學位類別: 博士
Doctor
系所名稱: 工學院 - 奈米工程與微系統研究所
Institute of NanoEngineering and MicroSystems
論文出版年: 2015
畢業學年度: 103
語文別: 英文
論文頁數: 66
中文關鍵詞: 實驗室晶片肺部發炎細胞遷移
外文關鍵詞: Lab on a chip, lung inflammation, cell migration
相關次數: 點閱:3下載:0
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 項研究報告指出一個仿生微系統,其重建成了肺部微環境監測對於肺部炎症嗜酸性白血球陽離子蛋白(ECP)的作用。ECP誘導氣管上皮細胞釋放CXCL-12,反過來又刺激上皮纖維細胞的往氣管上皮細胞遷移。這兩層微流體系統提供了用於灌注培養一個可行的擴散平台,並且被用於本研究顯示,CXCL12-CXCR4為主軸介導的ECP誘導纖維細胞外滲在肺部炎症。
    這個'肺上的實驗室“微型裝置可當作是動態的Transwell系統,透過導入流體可以重建血管-組織界面的體外測定,提高臨床前研究用。我們嘗試開發一種新的微流體模型不僅模擬Transwell的細胞遷移,但它也同時可以研究模仿生理條件在體內的遷移。由於血管是我們身體的一部分,這種模式使得研究在涉及血管於器官移植在體外模型更加逼真的機會。


    This study reports a biomimetic microsystem which reconstitutes the lung microenvironment for monitoring the role of eosinophil cationic protein (ECP) in lung inflammation. ECP induces airway epithelial cell expression of CXCL-12, which in turn stimulates the migration of fibrocytes towards the epithelium. This two-layered microfluidic system provides a feasible platform for perfusion culture, and was used in this study to reveal that the CXCL12-CXCR4 axis mediates ECP induced fibrocyte extravasation in lung inflammation.
    This ‘lung-on-a-chip’ microdevice serves as a dynamic transwell system by introducing flow that can reconstitute the blood vessel-tissue interface for in vitro assays, enhancing pre-clinical studies. We made an attempt to develop a new microfluidic model which not only simulates the transwell for studying cell migration, but it could also study the migration in the presence of flow mimicking the physiological conditions in the body. As blood vessels are the integral part of our body, this model gives an opportunity to study more realistic in vitro models of organs where blood vessel i.e. flow based migration is involved.

    Abstract 2 Acknowledgements 3 Abbreviations 4 LIST OF FIGURES 5 LIST OF TABLES 9 CHAPTER 1 13 INTRODUCTION 13 1.1 Background 13 1.2 Chemotaxis 13 1.3 Airway inflammation and fibrocyte infiltration 20 CHAPTER 2 23 MATERIALS AND METHODS 23 2.1 Cell culture 23 2.2 Purification of circulating fibrocytes 23 2.3 Recombinant ECP 24 2.4 RT-PCR and quantitative real-time PCR 25 2.5 Human CXCL-12 enzyme-link immunosorbent assay (ELISA) 26 2.6 Transwell assay 26 CHAPTER 3 27 DEVICE DESIGN AND FABRICATION 27 3.1 Device design 27 3.2. Device fabrication 28 CHAPTER 4 33 EXPERIMENTAL PROCESS 33 4.1 On chip cell culture and fibrocyte migration 33 4.2 Traditional migration assay 34 CHAPTER 5 35 RESULTS AND DISCUSSION 35 5.1 ECP-6His effects on mRNA and protein expression of CXCL-12 in Beas-2B cells 35 5.2 Fibrocyte migration towards ECP-6His-stimulated Beas-2B cells in the transwell system 37 5.3 Fibrocyte transmigration towards Beas-2B cells treated with ECP-6His on the lung-on-a-chip device 50 CHAPTER 6 60 CONCLUSIONS AND FUTURE WORK 60 6.1 Conclusions 60 6.2 Future Work 62 REFERENCES 64

    1. Peng, H. and E.L. Herzog, Fibrocytes: emerging effector cells in chronic inflammation. Current Opinion in Pharmacology, 2012. 12(4): p. 491-496.
    2. Huh, D., et al., Microengineered physiological biomimicry: organs-on-chips. Lab Chip, 2012. 12(12): p. 2156-64.
    3. Sung, J.H. and M.L. Shuler, Microtechnology for mimicking in vivo tissue environment. Ann Biomed Eng, 2012. 40(6): p. 1289-300.
    4. van der Meer, A.D. and A. van den Berg, Organs-on-chips: breaking the in vitro impasse. Integr Biol, 2012. 4(5): p. 461-70.
    5. Ho, C.-T., et al., Rapid heterogeneous liver-cell on-chip patterning via the enhanced field-induced dielectrophoresis trap. Lab on a Chip, 2006. 6(6): p. 724-734.
    6. Lee, S.-A., et al., Spheroid-based three-dimensional liver-on-a-chip to investigate hepatocyte-hepatic stellate cell interactions and flow effects. Lab on a Chip, 2013. 13(18): p. 3529-3537.
    7. Khetani, S.R. and S.N. Bhatia, Microscale culture of human liver cells for drug development. Nat Biotech, 2008. 26(1): p. 120-126.
    8. Ho, C.T., et al., Liver-cell patterning Lab Chip: mimicking the morphology of liver lobule tissue. Lab Chip, 2013. 13(18): p. 3578-87.
    9. Franco, C. and H. Gerhardt, Tissue engineering: Blood vessels on a chip. Nature, 2012. 488(7412): p. 465-466.
    10. Moya, M.L., et al., In Vitro Perfused Human Capillary Networks. Tissue Engineering Part C-Methods, 2013. 19(9): p. 730-737.
    11. Jang, K.-J., et al., Human kidney proximal tubule-on-a-chip for drug transport and nephrotoxicity assessment. Integrative Biology, 2013. 5(9): p. 1119-1129.
    12. Baudoin, R., et al., Development of a renal microchip for in vitro distal tubule models. Biotechnol Prog, 2007. 23(5): p. 1245-53.
    13. Jang, K., et al., Development of an osteoblast-based 3D continuous-perfusion microfluidic system for drug screening. Analytical and Bioanalytical Chemistry, 2008. 390(3): p. 825-832.
    14. Altmann, B., et al., Promotion of osteoblast differentiation in 3D biomaterial micro-chip arrays comprising fibronectin-coated poly(methyl methacrylate) polycarbonate. Biomaterials, 2011. 32(34): p. 8947-8956.
    15. Skolimowski, M., et al., Modular microfluidic system as a model of cystic fibrosis airways. Biomicrofluidics, 2012. 6(3): p. 034109-11.
    16. Huh, D., et al., Reconstituting Organ-Level Lung Functions on a Chip. Science, 2010. 328(5986): p. 1662-1668.
    17. Harris, S. and M. Shuler, Growth of endothelial cells on microfabricated silicon nitride membranes for anin vitro model of the blood-brain barrier. Biotechnology and Bioprocess Engineering, 2003. 8(4): p. 246-251.
    18. Griep, L.M., et al., BBB on chip: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function. Biomed Microdevices, 2013. 15(1): p. 145-50.
    19. Millet, L.J. and M.U. Gillette, New perspectives on neuronal development via microfluidic environments. Trends in Neurosciences, 2012. 35(12): p. 752-761.
    20. Kim, H.J. and D.E. Ingber, Gut-on-a-Chip microenvironment induces human intestinal cells to undergo villus differentiation. Integrative Biology, 2013. 5(9): p. 1130-40.
    21. Kim, H.J., et al., Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab on a Chip, 2012. 12(12): p. 2165-2174.
    22. Knight, D., Epithelium-fibroblast interactions in response to airway inflammation. Immunol Cell Biol, 2001. 79(2): p. 160-164.
    23. Bousquet, J., et al., Eosinophilic Inflammation in Asthma. New England Journal of Medicine, 1990. 323(15): p. 1033-1039.
    24. Vicente-Manzanares, M., D.J. Webb, and A.R. Horwitz, Cell migration at a glance. J Cell Sci, 2005. 118(Pt 21): p. 4917-9.
    25. Berzat, A. and A. Hall, Cellular responses to extracellular guidance cues. The EMBO Journal, 2010. 29(16): p. 2734-2745.
    26. Bagorda, A. and C.A. Parent, Eukaryotic chemotaxis at a glance. Journal of Cell Science, 2008. 121(16): p. 2621-2624.
    27. Stadelmann, W.K., A.G. Digenis, and G.R. Tobin, Physiology and healing dynamics of chronic cutaneous wounds. The American Journal of Surgery, 1998. 176(2, Supplement 1): p. 26S-38S.
    28. Mantovani, A., et al., Cancer-related inflammation. Nature, 2008. 454(7203): p. 436-444.
    29. Hillen, F. and A. Griffioen, Tumour vascularization: sprouting angiogenesis and beyond. Cancer and Metastasis Reviews, 2007. 26(3): p. 489-502.
    30. Davignon, J. and P. Ganz, Role of Endothelial Dysfunction in Atherosclerosis. Circulation, 2004. 109(23 suppl 1): p. III-27-III-32.
    31. Sitia, S., et al., From endothelial dysfunction to atherosclerosis. Autoimmunity Reviews, 2010. 9(12): p. 830-834.
    32. Behar, T., et al., GABA-induced chemokinesis and NGF-induced chemotaxis of embryonic spinal cord neurons. The Journal of Neuroscience, 1994. 14(1): p. 29-38.
    33. Ley, K., et al., Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol, 2007. 7(9): p. 678-689.
    34. Cinamon, G., et al., Chemoattractant Signals and β2 Integrin Occupancy at Apical Endothelial Contacts Combine with Shear Stress Signals to Promote Transendothelial Neutrophil Migration. The Journal of Immunology, 2004. 173(12): p. 7282-7291.
    35. Boyden, S., The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. The Journal of Experimental Medicine, 1962. 115(3): p. 453-466.
    36. Nelson, R.D., P.G. Quie, and R.L. Simmons, Chemotaxis Under Agarose: A New and Simple Method for Measuring Chemotaxis and Spontaneous Migration of Human Polymorphonuclear Leukocytes and Monocytes. The Journal of Immunology, 1975. 115(6): p. 1650-1656.
    37. Zheng, J.Q., et al., Turning of nerve growth cones induced by neurotransmitters. Nature, 1994. 368(6467): p. 140-144.
    38. Zigmond, S., Ability of polymophonuclear leukocytes to orient in gradients of chemotactic factors. J Cell Biol, 1977. 75: p. 606 - 616.
    39. Zicha, D., G. Dunn, and G. Jones, Analyzing Chemotaxis Using the Dunn Direct-Viewing Chamber. 1997. p. 449-457.
    40. Rebuck, J.W. and J.H. Crowley, A METHOD OF STUDYING LEUKOCYTIC FUNCTIONS IN VIVO. Annals of the New York Academy of Sciences, 1955. 59(5): p. 757-805.
    41. Czarnetzki, B.M., et al., Effect of neutrophil-derived eosinophil chemotactic factor (ECF) in human and guinea pig skin. J Invest Dermatol, 1980. 74(2): p. 109-11.
    42. Czarnetzki, B.M., et al., Chemotaxis: Basic aspects of methodology, mechanisms and pathology. Archives of Dermatological Research, 1983. 275(6): p. 359-364.
    43. Boyden, S., The chemotatic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes. J Exp Med, 1962. 115: p. 453 - 466.
    44. Sorg, C., et al., Use of a multiwell assembly for chemotaxis and evaluation by enzyme-linked immunosorbent assay (ELISA). Immunobiology, 1982. 162(2): p. 192-8.
    45. Nelson, R., P. Quie, and R. Simmons, Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J Immunol, 1975. 115: p. 1650 - 1656.
    46. Lohof, A.M., et al., Asymmetric modulation of cytosolic cAMP activity induces growth cone turning. J Neurosci, 1992. 12(4): p. 1253-61.
    47. Lin, F. and E.C. Butcher, T cell chemotaxis in a simple microfluidic device. Lab Chip, 2006. 6(11): p. 1462-9.
    48. Lin, F., et al., Neutrophil migration in opposing chemoattractant gradients using microfluidic chemotaxis devices. Ann Biomed Eng, 2005. 33(4): p. 475-82.
    49. Saadi, W., et al., A parallel-gradient microfluidic chamber for quantitative analysis of breast cancer cell chemotaxis. Biomed Microdevices, 2006. 8: p. 109 - 118.
    50. Lin, F., et al., Effective neutrophil chemotaxis is strongly influenced by mean IL-8 concentration. Biochem Biophys Res Commun, 2004. 319: p. 576 - 581.
    51. Chung, S., et al., Cell migration into scaffolds under co-culture conditions in a microfluidic platform. Lab on a Chip, 2009. 9(2): p. 269-275.
    52. Chen, Z., et al., In Vitro Model on Glass Surfaces for Complex Interactions between Different Types of Cells. Langmuir, 2010. 26(23): p. 17790-17794.
    53. Abhyankar, V.V., et al., Characterization of a membrane-based gradient generator for use in cell-signaling studies. Lab on a Chip, 2006. 6(3): p. 389-393.
    54. Chung, B.G., F. Lin, and N.L. Jeon, A microfluidic multi-injector for gradient generation. Lab on a Chip, 2006. 6(6): p. 764-768.
    55. Huang, C.P., et al., Engineering microscale cellular niches for three-dimensional multicellular co-cultures. Lab on a Chip, 2009. 9(12): p. 1740-1748.
    56. Ambravaneswaran, V., et al., Directional decisions during neutrophil chemotaxis inside bifurcating channels. Integrative Biology, 2010. 2(11-12): p. 639-647.
    57. Brightling, C.E., et al., The CXCL10/CXCR3 Axis Mediates Human Lung Mast Cell Migration to Asthmatic Airway Smooth Muscle. American Journal of Respiratory and Critical Care Medicine, 2005. 171(10): p. 1103-1108.
    58. Phillips, R.J., et al., Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest, 2004. 114(3): p. 438-46.
    59. Corrigan, C., Mechanisms of asthma. Medicine, 2008. 36(4): p. 177-180.
    60. Wilson, J.W. and T.L. Bamford, Assessing the Evidence for Remodelling of the Airway in Asthma. Pulmonary Pharmacology & Therapeutics, 2001. 14(3): p. 229-247.
    61. Humbles, A.A., et al., A Critical Role for Eosinophils in Allergic Airways Remodeling. Science, 2004. 305(5691): p. 1776-1779.
    62. Gleich, G.J., Mechanisms of eosinophil-associated inflammation. J Allergy Clin Immunol, 2000. 105(4): p. 651-63.
    63. Hohlfeld, J.M., et al., Eosinophil cationic protein alters pulmonary surfactant structure and function in asthma. Journal of Allergy and Clinical Immunology, 2004. 113(3): p. 496-502.
    64. Saunders, R., et al., Fibrocyte localization to the airway smooth muscle is a feature of asthma. Journal of Allergy and Clinical Immunology, 2009. 123(2): p. 376-384.
    65. Wang, C.H., et al., Increased circulating fibrocytes in asthma with chronic airflow obstruction. Am J Respir Crit Care Med, 2008. 178(6): p. 583-91.
    66. Nihlberg, K., et al., Tissue fibrocytes in patients with mild asthma: a possible link to thickness of reticular basement membrane? Respir Res, 2006. 7: p. 50.
    67. Reilkoff, R.A., R. Bucala, and E.L. Herzog, Fibrocytes: emerging effector cells in chronic inflammation. Nat Rev Immunol, 2011. 11(6): p. 427-435.

    無法下載圖示 全文公開日期 本全文未授權公開 (校內網路)
    全文公開日期 本全文未授權公開 (校外網路)
    全文公開日期 本全文未授權公開 (國家圖書館:臺灣博碩士論文系統)
    QR CODE