臺灣博碩士論文加值系統

細胞在生物體內的生長發育、分化和移動除了受到生物化學刺激之外，物理性刺激如細胞外基質硬度以及滲透壓亦對細胞行為有很大影響。在我們的實驗當中，我們將H9C2肌肉母細胞培養在不同硬度細胞外基質上並且分成對照組和水壓組來探討此肌肉母細胞同時受細胞外基質軟硬度和靜水壓的刺激對細胞分化和細胞型態的影響。另一方面，我們也探討T細胞在二維和三維細胞培養環境微流道中在均勻和梯度滲透壓的情況下，細胞的遷移是否會受到影響。
我們使用聚丙烯醯胺(Polyacrylamide, PA)水膠來當作細胞外基質材料，因為PA水膠的硬度能夠透過調配不同比例的acrylamide 和bis-acrylamide 來製作出不同軟硬度的水膠。H9C2細胞培養於蛋白質fibronectin 附著的水膠表面再分成控制組和10公分靜水壓組並培養48小時後，觀測細胞分化採轉譯因子MyoD表現量和細胞型態。實驗結果顯示，MyoD的表現量在水壓組皆高於控制組，而細胞面積在經過水壓刺激後亦會較控制組來的大，但細胞長寬比則較無明顯差異。因此，靜水壓可能為促進H9C2細胞分化和增加細胞面積的其中一種刺激因素。
在細胞遷移實驗方面，我們將T細胞培養於三種不同滲透壓培養液內(260、337、以及415 mOsm)並計算細胞移動速度。初步實驗結果顯示T細胞在低滲透壓的細胞培養液中，其移動速度會較慢。此外，我們亦製作出兩種微流道來產生滲透壓梯度以觀察T細胞是否會受到滲透壓梯度刺激而影響細胞遷移行為。此兩種裝置分別為二維細胞培養環境的agarose-based微流道和三維細胞培養環境的collagen-filled微流道。滲透壓梯度於三維細胞培養環境微流道能產生約0.031mM/μm的滲透壓梯度，而T細胞在這兩種流道裝置內分別給予均勻和梯度滲透壓環境來觀察細胞的遷移行為。在有滲透壓濃度梯度的環境中並無觀察到細胞會有特定的遷移趨向性。

The cell growth, proliferation, differentiation, and migration are affected by biochemical and biophysical cues, such as the stiffness of extracellular matrix (ECM) and osmolarity. In this work, we studied the effects of hydrostatic pressure and substrate stiffness on the differentiation and morphology of H9C2 myoblast. We also investigated the effects of homogeneous and gradient osmolarity on T cell migration using 2D and 3D microfluidic devices. Polyacrylamide (PA) hydrogel was used as cell culturing substrates and the stiffness of the PA gel was modulated by mixing different ratios of acrylamide to bis-acrylamide. The cell size, aspect ratio, and expression of transcription factor MyoD were measured after culturing the H9C2 cell on fibronectin coated PA gel with and without the presence of 10 cm hydrostatic pressure for 48 hours. The results reveal that the 10-cm-hydrostatic- pressure exposure increased the MyoD expression and cell size, but resulted in insignificant shape change when compared with cells cultured without presence of the pressure. These findings indicate that the hydrostatic pressure promotes the MyoD expression and increases the cell area. As for the cell migration studies, we cultured QL-9 T cells in medium of osmolarity 260, 337, and 415 mOsm. Live cell images were acquired and analyzing the images revealed that the hypotonic solution significantly decreased the migration speed of the T cells. Two microfluidic-based devices were herein designed for studying the cell migration in response to osmotic gradient: one was a three-chambers design made of agarose for studying the cell migration in a 2D plane; another was a two-chambers device with a collagen-filled tunnel in between for investigating cell movement in a 3D space. The osmotic gradient in the collagen-filled tunnel was about 0.031mOsm/μm. Preliminary results showed that such an osmotic gradient did not evoke gradient-directed movement in T cells.

口試委員會審定書 #
誌謝 i
中文摘要 ii
ABSTRACT iii
CONTENTS v
LIST OF FIGURES viii
LIST OF TABLES xi
Chapter 1 Introduction 1
1.1 Hydrostatic Pressure and ECM Stiffness influence on H9C2 Cell Morphology and Differentiation 1
1.1.1 The Stiffness of Extracellular Matrix 2
1.1.2 Hydrostatic Pressure 4
1.1.3 Previous Study in our Lab 5
1.1.4 Purpose of the Study 8
1.2 Osmolarity Influences on T Cell Migration 9
1.2.1 Osmolarity 9
1.2.2 Aquaporin1 (AQP1) 10
1.2.3 Purpose of the Study 10
Chapter 2 Materials and Method 12
2.1 Hydrostatic Pressure Experiment 12
2.1.1 Coverslips Surface Modification 12
2.1.2 PA (polyacrylamide) Hydrogels Fabrication 13
2.1.3 Functionalization of PA Hydrogels 15
2.1.4 H9C2 Cell Culture and Hydrostatic Pressure Experiment 17
2.1.5 H9C2 Cells Treated with Rho Inhibitor 17
2.1.6 Immunofluorescence staining 18
2.1.7 Cell Image Capturing and Analyzing 19
2.2 T Cell Cultured in Medium with Different Osmolarity 20
2.2.1 T Cell Preparation 20
2.2.2 Preparation of Medium with Different Osmolarity 20
2.2.3 T Cell Culturing and T Cell Image Analyzing 21
2.2.4 Aquaporin1 (AQP1) Immunofluorescence staining 21
2.3 T Cell Migration in Osmotic Gradient Microfluidic Devices 22
2.3.1 Fabrication of Agarose-based Microfluidic (2D) Device 23
2.3.2 Photolithography of Master Mold for Collagen-filled Microfluidic (3D) Device 25
2.3.3 Soft lithography 26
2.3.4 Preparation of Collagen Solution 27
2.3.5 Integration of Collagen-filled Microfluidic Device 28
2.3.6 Quantification of the Concentration Gradient 29
2.3.7 T Cell Cultured in Collagen-Filled Microfluidics with Osmotic Gradient 29
Chapter 3 Result and Discussion 30
3.1 Fibronectin Density of Different Elastic Hydrogels 30
3.2 H9C2 on Different Elastic Hydrogels under Hydrostatic Pressure 31
3.2.1 Morphological findings of H9C2 Cell 31
3.2.2 H9C2 Cell Area 33
3.2.3 H9C2 Cell Aspect Ratio 34
3.2.4 MyoD and Myogenin Expression 36
3.2.5 Rho Inhibitor Treated H9C2 on 10 kPa Hydrogels 40
3.2.6 Discussion of H9C2 Cell on Different Stiffness Substrates with and without Pressure 41
3.3 T Cell in Medium with Different Osmolarity 42
3.3.1 T Cell Migration in Medium with Different Osmolarity 42
3.3.2 Aquaporin1 Expression of T Cell in Medium with Different Osmolarity 43
3.4 Concentration Generation and Analysis 44
3.4.1 Typan Blue in the Agarose-Based Microfluidics 44
3.4.2 Trypan Blue and Cy3 Gradient in the Collagen-Filled Microfluidics 45
3.5 T Cell Migration in Microfluidic Devices 46
3.5.1 T Cell in the Agarose-Based Microfluidics 46
3.5.2 T Cell Migration in Collagen-Filled Microfluidics 47
Chapter 4 Conclusion and Future Works 49
References 51

[1]D. E. Jaalouk and J. Lammerding, "Mechanotransduction gone awry," Nature reviews Molecular cell biology, vol. 10, pp. 63-73, 2009.
[2]T. Mammoto and D. E. Ingber, "Mechanical control of tissue and organ development," Development, vol. 137, pp. 1407-1420, 2010.
[3]P. A. Janmey and R. T. Miller, "Mechanisms of mechanical signaling in development and disease," Journal of cell science, vol. 124, pp. 9-18, 2011.
[4]I. Kratchmarova, B. Blagoev, M. Haack-Sorensen, M. Kassem, and M. Mann, "Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation," Science Signaling, vol. 308, p. 1472, 2005.
[5]A. J. Keung, S. Kumar, and D. V. Schaffer, "Presentation counts: microenvironmental regulation of stem cells by biophysical and material cues," Annual review of cell and developmental biology, vol. 26, pp. 533-556, 2010.
[6]S. F. Badylak, "Regenerative medicine and developmental biology: the role of the extracellular matrix," The Anatomical Record Part B: The New Anatomist, vol. 287, pp. 36-41, 2005.
[7]K. S. Kolahi and M. R. Mofrad, "Mechanotransduction: a major regulator of homeostasis and development," Wiley Interdisciplinary Reviews: Systems Biology and Medicine, vol. 2, pp. 625-639, 2010.
[8]H.-B. Wang, M. Dembo, and Y.-L. Wang, "Substrate flexibility regulates growth and apoptosis of normal but not transformed cells," American Journal of Physiology-Cell Physiology, vol. 279, pp. C1345-C1350, 2000.
[9]C. A. Reinhart-King, M. Dembo, and D. A. Hammer, "The dynamics and mechanics of endothelial cell spreading," Biophysical journal, vol. 89, pp. 676-689, 2005.
[10]R. J. Pelham and Y.-l. Wang, "Cell locomotion and focal adhesions are regulated by substrate flexibility," Proceedings of the National Academy of Sciences, vol. 94, pp. 13661-13665, 1997.
[11]L. A. Flanagan, Y.-E. Ju, B. Marg, M. Osterfield, and P. A. Janmey, "Neurite branching on deformable substrates," Neuroreport, vol. 13, p. 2411, 2002.
[12]D. E. Discher, P. Janmey, and Y.-l. Wang, "Tissue cells feel and respond to the stiffness of their substrate," Science, vol. 310, pp. 1139-1143, 2005.
[13]D. E. Discher, D. J. Mooney, and P. W. Zandstra, "Growth factors, matrices, and forces combine and control stem cells," Science, vol. 324, pp. 1673-1677, 2009.
[14]A. J. Engler, M. A. Griffin, S. Sen, C. G. Bonnemann, H. L. Sweeney, and D. E. Discher, "Myotubes differentiate optimally on substrates with tissue-like stiffness pathological implications for soft or stiff microenvironments," The Journal of cell biology, vol. 166, pp. 877-887, 2004.
[15]A. J. Engler, S. Sen, H. L. Sweeney, and D. E. Discher, "Matrix elasticity directs stem cell lineage specification," Cell, vol. 126, pp. 677-689, 2006.
[16]A. Gefen, N. Gefen, Q. Zhu, R. Raghupathi, and S. S. Margulies, "Age-dependent changes in material properties of the brain and braincase of the rat," Journal of neurotrauma, vol. 20, pp. 1163-1177, 2003.
[17]G. F. Mitchell, H. Parise, E. J. Benjamin, M. G. Larson, M. J. Keyes, J. A. Vita, et al., "Changes in arterial stiffness and wave reflection with advancing age in healthy men and women the Framingham Heart Study," Hypertension, vol. 43, pp. 1239-1245, 2004.
[18]A. J. Keung, K. E. Healy, S. Kumar, and D. V. Schaffer, "Biophysics and dynamics of natural and engineered stem cell microenvironments," Wiley Interdisciplinary Reviews: Systems Biology and Medicine, vol. 2, pp. 49-64, 2010.
[19]J. P. Winer, P. A. Janmey, M. E. McCormick, and M. Funaki, "Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli," Tissue Engineering Part A, vol. 15, pp. 147-154, 2008.
[20]S. X. Hsiong, P. Carampin, H. J. Kong, K. Y. Lee, and D. J. Mooney, "Differentiation stage alters matrix control of stem cells," Journal of Biomedical Materials Research Part A, vol. 85, pp. 145-156, 2008.
[21]A. J. Engler, C. Carag-Krieger, C. P. Johnson, M. Raab, H.-Y. Tang, D. W. Speicher, et al., "Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating," Journal of cell science, vol. 121, pp. 3794-3802, 2008.
[22]A. Engler, L. Bacakova, C. Newman, A. Hategan, M. Griffin, and D. Discher, "Substrate compliance versus ligand density in cell on gel responses," Biophysical journal, vol. 86, pp. 617-628, 2004.
[23]P. C. Georges and P. A. Janmey, "Cell type-specific response to growth on soft materials," Journal of Applied Physiology, vol. 98, pp. 1547-1553, 2005.
[24]C.-M. Lo, H.-B. Wang, M. Dembo, and Y.-l. Wang, "Cell movement is guided by the rigidity of the substrate," Biophysical journal, vol. 79, pp. 144-152, 2000.
[25]F. Guilak, D. M. Cohen, B. T. Estes, J. M. Gimble, W. Liedtke, and C. S. Chen, "Control of stem cell fate by physical interactions with the extracellular matrix," Cell stem cell, vol. 5, p. 17, 2009.
[26]A. Agar, S. S. Yip, M. A. Hill, and M. T. Coroneo, "Pressure related apoptosis in neuronal cell lines," Journal of neuroscience research, vol. 60, pp. 495-503, 2000.
[27]R. M. Sappington, M. Chan, and D. J. Calkins, "Interleukin-6 protects retinal ganglion cells from pressure-induced death," Investigative ophthalmology & visual science, vol. 47, pp. 2932-2942, 2006.
[28]M. R. Drumm, B. D. York, and J. Nagatomi, "Effect of sustained hydrostatic pressure on rat bladder smooth muscle cell function," Urology, vol. 75, pp. 879-885, 2010.
[29]D.-x. Luo, J. Cheng, Y. Xiong, J. Li, C. Xia, C. Xu, et al., "Static pressure drives proliferation of vascular smooth muscle cells via caveolin-1/ERK1/2 pathway," Biochemical and biophysical research communications, vol. 391, pp. 1693-1697, 2010.
[30]B. E. Sumpio, M. D. Widmann, J. Ricotta, M. A. Awolesi, and M. Watase, "Increased ambient pressure stimulates proliferation and morphologic changes in cultured endothelial cells," Journal of cellular physiology, vol. 158, pp. 133-139, 1994.
[31]E. A. Schwartz, R. Bizios, M. S. Medow, and M. E. Gerritsen, "Exposure of Human Vascular Endothelial Cells to Sustained Hydrostatic Pressure Stimulates Proliferation Involvement of the αV Integrins," Circulation research, vol. 84, pp. 315-322, 1999.
[32]N. Onoue, J. Nawata, T. Tada, D. Zhulanqiqige, H. Wang, K. Sugimura, et al., "Increased static pressure promotes migration of vascular smooth muscle cells: involvement of the Rho-kinase pathway," Journal of cardiovascular pharmacology, vol. 51, pp. 55-61, 2008.
[33]B. Bourns, S. Franklin, L. Cassimeris, and E. Salmon, "High hydrostatic pressure effects in vivo: changes in cell morphology, microtubule assembly, and actin organization," Cell motility and the cytoskeleton, vol. 10, pp. 380-390, 1988.
[34]J. Liu, Z. Zhao, J. Li, L. Zou, C. Shuler, Y. Zou, et al., "Hydrostatic pressures promote initial osteodifferentiation with ERK1/2 not p38 MAPK signaling involved," Journal of cellular biochemistry, vol. 107, pp. 224-232, 2009.
[35]D. R. Wagner, D. P. Lindsey, K. W. Li, P. Tummala, S. E. Chandran, R. L. Smith, et al., "Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium," Annals of biomedical engineering, vol. 36, pp. 813-820, 2008.
[36]P. Angele, J. Yoo, C. Smith, J. Mansour, K. Jepsen, M. Nerlich, et al., "Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro," Journal of orthopaedic research, vol. 21, pp. 451-457, 2003.
[37]R. Ogawa, S. Mizuno, G. F. Murphy, and D. P. Orgill, "The Effect of Hydrostatic Pressure on Three-Dimensional Chondroinduction of Human Adipose–Derived Stem Cells," Tissue Engineering Part A, vol. 15, pp. 2937-2945, 2009.
[38]B. D. Elder and K. A. Athanasiou, "Hydrostatic pressure in articular cartilage tissue engineering: from chondrocytes to tissue regeneration," Tissue Engineering Part B: Reviews, vol. 15, pp. 43-53, 2009.
[39]J. Arnadottir and M. Chalfie, "Eukaryotic mechanosensitive channels," Annual review of biophysics, vol. 39, pp. 111-137, 2010.
[40]L. H. Romer, K. G. Birukov, and J. G. Garcia, "Focal adhesions paradigm for a signaling nexus," Circulation research, vol. 98, pp. 606-616, 2006.
[41]A. Liedert, D. Kaspar, R. Blakytny, L. Claes, and A. Ignatius, "Signal transduction pathways involved in mechanotransduction in bone cells," Biochemical and biophysical research communications, vol. 349, pp. 1-5, 2006.
[42]M. H. Parker, P. Seale, and M. A. Rudnicki, "Looking back to the embryo: defining transcriptional networks in adult myogenesis," Nature reviews genetics, vol. 4, pp. 497-507, 2003.
[43]T. Ohashi, Y. Sugaya, N. Sakamoto, and M. Sato, "Hydrostatic pressure influences morphology and expression of VE-cadherin of vascular endothelial cells," Journal of biomechanics, vol. 40, pp. 2399-2405, 2007.
[44]X.-D. Ren, W. B. Kiosses, and M. A. Schwartz, "Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton," The EMBO Journal, vol. 18, pp. 578-585, 1999.
[45]H. A. Benink and W. M. Bement, "Concentric zones of active RhoA and Cdc42 around single cell wounds," The Journal of cell biology, vol. 168, pp. 429-439, 2005.
[46]A. Burakov, E. Nadezhdina, B. Slepchenko, and V. Rodionov, "Centrosome positioning in interphase cells," The Journal of cell biology, vol. 162, pp. 963-969, 2003.
[47]M. L. Gardel, I. C. Schneider, Y. Aratyn-Schaus, and C. M. Waterman, "Mechanical integration of actin and adhesion dynamics in cell migration," Annual review of cell and developmental biology, vol. 26, pp. 315-333, 2010.
[48]A. J. Ridley, M. A. Schwartz, K. Burridge, R. A. Firtel, M. H. Ginsberg, G. Borisy, et al., "Cell migration: integrating signals from front to back," Science, vol. 302, pp. 1704-1709, 2003.
[49]D. M. Rose, R. Alon, and M. H. Ginsberg, "Integrin modulation and signaling in leukocyte adhesion and migration," Immunological reviews, vol. 218, pp. 126-134, 2007.
[50]S.-J. Wang, W. Saadi, F. Lin, C. Minh-Canh Nguyen, and N. Li Jeon, "Differential effects of EGF gradient profiles on MDA-MB-231 breast cancer cell chemotaxis," Experimental cell research, vol. 300, pp. 180-189, 2004.
[51]N. L. Jeon, H. Baskaran, S. K. Dertinger, G. M. Whitesides, L. Van De Water, and M. Toner, "Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device," Nature biotechnology, vol. 20, pp. 826-830, 2002.
[52]P. Fauchald, "Transcapillary colloid osmotic gradient and body fluid volumes in renal failure," Kidney international, vol. 29, pp. 895-900, 1986.
[53]S. Hatashita, J. T. Hoff, and S. M. Salamat, "Ischemic brain edema and the osmotic gradient between blood and brain," Journal of Cerebral Blood Flow & Metabolism, vol. 8, pp. 552-559, 1988.
[54]L. Yeyang, L. Xilin, and L. Yi, "Changes in serum osmolality and osmolar discrepancy in burned patients," Burns, vol. 18, pp. 22-25, 1992.
[55]S. Saadoun, M. C. Papadopoulos, H. Watanabe, D. Yan, G. T. Manley, and A. Verkman, "Involvement of aquaporin-4 in astroglial cell migration and glial scar formation," Journal of cell science, vol. 118, pp. 5691-5698, 2005.
[56]V. M. Loitto, T. Karlsson, and K. E. Magnusson, "Water flux in cell motility: expanding the mechanisms of membrane protrusion," Cell motility and the cytoskeleton, vol. 66, pp. 237-247, 2009.
[57]A. Verkman, "More than just water channels: unexpected cellular roles of aquaporins," Journal of cell science, vol. 118, pp. 3225-3232, 2005.
[58]M. Papadopoulos, S. Saadoun, and A. Verkman, "Aquaporins and cell migration," Pflugers Archiv-European Journal of Physiology, vol. 456, pp. 693-700, 2008.
[59]S. Saadoun, M. C. Papadopoulos, M. Hara-Chikuma, and A. Verkman, "Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption," Nature, vol. 434, pp. 786-792, 2005.
[60]V. Falanga, "Wound healing and its impairment in the diabetic foot," The Lancet, vol. 366, pp. 1736-1743, 2005.
[61]J. E. Park and A. Barbul, "Understanding the role of immune regulation in wound healing," The American journal of surgery, vol. 187, pp. S11-S16, 2004.
[62]E. Engelhardt, A. Toksoy, M. Goebeler, S. Debus, E.-B. Brocker, and R. Gillitzer, "Chemokines IL-8, GROα, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing," The American journal of pathology, vol. 153, pp. 1849-1860, 1998.
[63]C. E. Kandow, P. C. Georges, P. A. Janmey, and K. A. Beningo, "Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses," Methods in cell biology, vol. 83, pp. 29-46, 2007.
[64]J. Y. Wong, A. Velasco, P. Rajagopalan, and Q. Pham, "Directed movement of vascular smooth muscle cells on gradient-compliant hydrogels," Langmuir, vol. 19, pp. 1908-1913, 2003.
[65]J. R. Tse and A. J. Engler, "Preparation of hydrogel substrates with tunable mechanical properties," Current protocols in cell biology, pp. 10.16. 1-10.16. 16, 2010.
[66]P. P. Provenzano, D. R. Inman, K. W. Eliceiri, S. M. Trier, and P. J. Keely, "Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization," Biophysical journal, vol. 95, pp. 5374-5384, 2008.
[67]R. R. Alfieri, M. A. Bonelli, A. Cavazzoni, M. Brigotti, C. Fumarola, P. Sestili, et al., "Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress," The Journal of physiology, vol. 576, pp. 391-401, 2006.
[68]K. Ren, L. Fourel, C. G. Rouviere, C. Albiges-Rizo, and C. Picart, "Manipulation of the adhesive behaviour of skeletal muscle cells on soft and stiff polyelectrolyte multilayers," Acta biomaterialia, vol. 6, pp. 4238-4248, 2010.
[69]T. Boontheekul, E. E. Hill, H.-J. Kong, and D. J. Mooney, "Regulating myoblast phenotype through controlled gel stiffness and degradation," Tissue engineering, vol. 13, pp. 1431-1442, 2007.
[70]T. Yeung, P. C. Georges, L. A. Flanagan, B. Marg, M. Ortiz, M. Funaki, et al., "Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion," Cell motility and the cytoskeleton, vol. 60, pp. 24-34, 2005.
[71]M. Ishido, K. Kami, and M. Masuhara, "Localization of MyoD, myogenin and cell cycle regulatory factors in hypertrophying rat skeletal muscles," Acta physiologica scandinavica, vol. 180, pp. 281-289, 2004.
[72]C. A. Lyssiotis, L. L. Lairson, A. E. Boitano, H. Wurdak, S. Zhu, and P. G. Schultz, "Chemical control of stem cell fate and developmental potential," Angewandte Chemie International Edition, vol. 50, pp. 200-242, 2011.