臺灣博碩士論文加值系統

細胞藉由黏著分子能夠與細胞和細胞外基質連結。黏著行為會受到細胞內部生理調控與外在環境的影響，對於細胞型態、移動、
運動和分化等生理方面及癌細胞轉移等病理方面皆扮演重要角色，
因此細胞的附著能力也意味著細胞執行正常生理行為的表現。目前有許多技術可用於量測細胞的附著力，如微吸管、光學鑷等，其中原子力顯微鏡有易於調控參數、生理環境下量測、高解析度等優勢，搭配使用細胞取代針尖功能的細胞探針技術，可量測細胞與基材、細胞與細胞之間的細胞附著力。基材的軟硬度被認為會影響細胞的行為，每種細胞對於軟硬度的感知與反應也不盡相同。
本研究欲探討短時間內基材軟硬度與細胞附著力的關係。實驗選用纖維母細胞，使用原子力顯微鏡搭配細胞探針技術去定量細胞附著力，細胞與基材的接觸時間定於30秒、60秒、90秒及300秒，所有基材表面塗佈有膠原蛋白第一型，硬度分成1037 Pa、10930 Pa及玻璃基材(控制組)，並且比較細胞附著力與接觸時間、基材硬度的關係。統計分析方法採用單因子變異數分析和涂基HSD檢定，
定義p<0.05有顯著差異。
實驗結果顯示在相同的接觸時間下，隨著基材硬度增加，細胞附著力有逐漸上升的趨勢，細胞附著力大小在1037 Pa、10930 Pa和玻璃基材，接觸時間30秒分別為296.99±107.73、459.69±228.51、
620.01 pN，接觸時間60秒分別為344.96±198.85、561.84±283.58、1917.3±344.28 pN，接觸時間90秒為347.44±125.22、644.75±358.57、2519.76±685.06 pN，接觸時間300秒分別為482.11±194.34、1820.11±949.29、3373.45±1867.02 pN，統計分析顯示三種基材硬度在各組的接觸時間的細胞附著力互相都有顯著差異。
另外，觀察三種硬度基材的細胞附著力與隨著時間增長的趨勢發現硬度1037 Pa的細胞附著力上升不明顯，硬度10930 Pa的細胞附著力在90秒到300秒之間有顯著上升，玻璃基材的細胞附著力在30秒至300秒之間呈現明顯上升且趨向穩定平衡的趨勢。
從結果發現細胞會受到基材軟硬度的影響而有不同表現，未來可以結合生化分析與螢光顯微鏡等技術，對於細胞內部黏著分子的分佈與變化有更多了解。

The cell can link another cell and ECM by adhesion molecules. The behavior of cell adhesion is influenced by the cell physiology and external environment. It plays an important role in cell morphology, motility, movement and differentiation, and cancer metastasis etc. So the ability to cell adhesion means that the cell carries out the normal physiological behavior. At present, a lot of technology can be used to quantify cell detaching force, for instance, micropipette, optical tweezers and so on. AFM has a lot of advantages, like parameters control easily, high resolution, and measuring in air. AFM combined with cell probe replacing tip function could measure the cell detaching force between cell-substrate and cell-cell. The rigidity of the substrates is considered to affect cell behavior. The rigidity sensitivity and respond of each cell is not the same.
The poupose of this study is to investigate the relation between the rigidity of substrate and cell detaching force. Fibroblast cell was used to quantify cell detaching force. The contact times were set at 30, 60, 90, and 300 seconds. All the surfaces of substrates were coated with collagen type Ⅰ. The rigidity of the substrates were 1037 Pa, 10930 Pa and glass (control group). Statistics is by one-way ANOVA and Tukey HSD.
Under the same contact time, the detaching force rise gradually with increasing substrate rigidity. Cell detaching force on 1037 Pa, 10930 Pa and glass substrates were 296.99±107.73, 459.69±228.51, and 620.01±211.98 pN respectively for 30 seconds contact time; the forces were 344.96±198.85, 561.84±283.58, and 1917.3±344.2 pN for 60 seconds; the forces were 347.44±125.22, 644.75±358.57, and 2519.76±685.06 pN for 90 seconds.; and the forces were 482.11±194.34, 1820.11±949.29, and 3373.45±1867.02 pN for 300 seconds. The cell detaching force among three rigidity substrates were significant different on each contact time.
The detaching force increased slowly on 1037 Pa, increasing notablely from 90 to 300 seconds on 10930 Pa, and increasing notablely to be stable from 30 to 300 seconds on glass.
Our results showed cell adhesion could be influenced by substrate rigidity. This cell probe technique can be further used for parameter study on cell substrate interaction in the future.

摘要 I
ABSTRACT III
誌謝 V
目次 VI
表目錄 IX
圖目錄 X
第1章 緒論 1
1.1 細胞黏著性 1
1.1.1 細胞與細胞外基質之交互作用 2
1.1.2 細胞黏著性之評估 5
1.2 評估細胞黏著力之研究與技術 7
1.2.1 微吸管 7
1.2.2 光學鑷 8
1.2.3 平行板流動系統 9
1.2.4 生物膜力探針 10
1.2.5 細胞刮取機構 11
1.2.6 原子力顯微鏡 12
1.3 原子力顯微鏡 13
1.3.1 基本原理與構造 13
1.3.2 影像量測 15
1.3.3 力學量測 16
1.4 細胞探針 18
1.5 基材軟硬度對細胞之影響 23
1.6 研究動機與架構 30
第2章 材料與方法 32
2.1 細胞培養 32
2.2 基材製備 33
2.3 原子力顯微鏡系統與探針 36
2.4 原子力顯微鏡探針功能化修飾 40
2.5 原子力顯微鏡探針校正與操作 41
2.6 細胞探針製作 42
2.7 細胞附著力量測 43
2.8 資料分析 45
2.8.1 力-距離曲線分析 45
2.8.2 統計分析 46
2.9 實驗流程 47
第3章 研究結果 48
3.1 細胞探針 48
3.2 細胞附著力與接觸時間 49
3.2.1 使用硬度1037 Pa膠體為基材的結果 49
3.2.2 使用硬度10930 Pa膠體為基材的結果 53
3.2.3 使用玻璃為基材的結果 (控制組) 57
3.3 細胞附著力與基材硬度 61
第4章 討論 68
4.1 實驗結果探討 68
4.1.1 細胞附著力的意義 69
4.1.2 細胞附著力與接觸時間 69
4.1.3 細胞附著力與基材硬度 73
4.1.4 力-距離曲線圖提供的資訊 75
4.2 實驗設計探討與限制 76
4.2.1 原子力顯微鏡探針的選用 76
4.2.2 細胞探針製作與使用的探討 77
4.2.3 基材製備與使用的探討 80
4.2.4 實驗量測環境的探討 81
4.2.5 細胞面積 81
4.3 未來展望 82
第5章 結論 84
參考文獻 86
自述 93

Arnaout MA, Goodman SL, Xiong JP. Structure and mechanics of integrin-based cell adhesion. Curr Opin Cell Biol 19 (5): 495-507, 2007.
Ashkin A, Dziedzic JM. Internal cell manipulation using infrared laser traps. Proc Natl Acad Sci U S A 86 (20): 7914-8, 1989.
Bausch AR, Moller W, Sackmann E. Measurement of local viscoelasticity and forces in living cells by magnetic tweezers. Biophys J 76 (1 Pt 1): 573-9, 1999.
Bausch AR, Ziemann F, Boulbitch AA, Jacobson K, Sackmann E. Local measurements of viscoelastic parameters of adherent cell surfaces by magnetic bead microrheometry. Biophys J 75 (4): 2038-49, 1998.
Benoit M. Cell adhesion measured by force spectroscopy on living cells. Methods Cell Biol 68: 91-114, 2002.
Bourdoulous S, Orend G, MacKenna DA, Pasqualini R, Ruoslahti E. Fibronectin matrix regulates activation of RHO and CDC42 GTPases and cell cycle progression. J Cell Biol 143 (1): 267-76, 1998.
Braun BC, Glickman M, Kraft R, Dahlmann B, Kloetzel PM, Finley D, Schmidt M. The base of the proteasome regulatory particle exhibits chaperone-like activity. Nat Cell Biol 1 (4): 221-6, 1999.
Butt HJ, Wolff EK, Gould SA, Dixon Northern B, Peterson CM, Hansma PK. Imaging cells with the atomic force microscope. J Struct Biol 105 (1-3): 54-61, 1990.
Calderwood DA. Integrin activation. J Cell Sci 117 (Pt 5): 657-66, 2004.
Chan BM, Kassner PD, Schiro JA, Byers HR, Kupper TS, Hemler ME. Distinct cellular functions mediated by different VLA integrin alpha subunit cytoplasmic domains. Cell 68 (6): 1051-60, 1992.
Charras GT, Horton MA. Determination of cellular strains by combined atomic force microscopy and finite element modeling. Biophys J 83 (2): 858-79, 2002.
Charras GT, Horton MA. Single cell mechanotransduction and its modulation analyzed by atomic force microscope indentation. Biophys J 82 (6): 2970-81, 2002.
Chu YS, Thomas WA, Eder O, Pincet F, Perez E, Thiery JP, Dufour S. Force measurements in E-cadherin-mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42. J Cell Biol 167 (6): 1183-94, 2004.
Discher DE, Janmey P, Wang YL. Tissue cells feel and respond to the stiffness of their substrate. Science 310 (5751): 1139-43, 2005.
Discher DE, Mohandas N, Evans EA. Molecular maps of red cell deformation: hidden elasticity and in situ connectivity. Science 266 (5187): 1032-5, 1994.
Engler AJ, Griffin MA, Sen S, Bonnemann CG, Sweeney HL, Discher DE. Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments. J Cell Biol 166 (6): 877-87, 2004.
Evans EA. Structure and deformation properties of red blood cells: concepts and quantitative methods. Methods Enzymol 173: 3-35, 1989.
Florin EL, Moy VT, Gaub HE. Adhesion forces between individual ligand-receptor pairs. Science 264 (5157): 415-7, 1994.
Florin EL, Pralle A, Horber JK, Stelzer EH. Photonic force microscope based on optical tweezers and two-photon excitation for biological applications. J Struct Biol 119 (2): 202-11, 1997.
Franz CM, Taubenberger A, Puech PH, Muller DJ. Studying integrin-mediated cell adhesion at the single-molecule level using AFM force spectroscopy. Sci STKE 2007 (406): pl5, 2007.
Gaboriaud F, Dufrene YF. Atomic force microscopy of microbial cells: application to nanomechanical properties, surface forces and molecular recognition forces. Colloids Surf B Biointerfaces 54 (1): 10-9, 2007.
Galbraith CG, Sheetz MP. A micromachined device provides a new bend on fibroblast traction forces. Proc Natl Acad Sci U S A 94 (17): 9114-8, 1997.
Galbraith CG, Yamada KM, Sheetz MP. The relationship between force and focal complex development. J Cell Biol 159 (4): 695-705, 2002.
Gallant ND, Garcia AJ. Model of integrin-mediated cell adhesion strengthening. J Biomech 40 (6): 1301-9, 2007.
Gallant ND, Michael KE, Garcia AJ. Cell adhesion strengthening: contributions of adhesive area, integrin binding, and focal adhesion assembly. Mol Biol Cell 16 (9): 4329-40, 2005.
Garcia AJ, Boettiger D. Integrin-fibronectin interactions at the cell-material interface: initial integrin binding and signaling. Biomaterials 20 (23-24): 2427-33, 1999.
Grandbois M, Beyer M, Rief M, Clausen-Schaumann H, Gaub HE. How strong is a covalent bond? Science 283 (5408): 1727-30, 1999.
Guo WH, Wang YL. Retrograde fluxes of focal adhesion proteins in response to cell migration and mechanical signals. Mol Biol Cell 18 (11): 4519-27, 2007.
Hall A. G proteins and small GTPases: distant relatives keep in touch. Science 280 (5372): 2074-5, 1998.
Hansma HG, Hoh JH. Biomolecular imaging with the atomic force microscope. Annu Rev Biophys Biomol Struct 23: 115-39, 1994.
Heinrich V, Leung A, Evans E. Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. II. Tether flow terminated by P-selectin dissociation from PSGL-1. Biophys J 88 (3): 2299-308, 2005.
Hoben G, Huang W, Thoma BS, LeBaron RG, Athanasiou KA. Quantification of varying adhesion levels in chondrocytes using the cytodetacher. Ann Biomed Eng 30 (5): 703-12, 2002.
Horber JK, Haberle W, Ohnesorge F, Binnig G, Liebich HG, Czerny CP, Mahnel H, Mayr A. Investigation of living cells in the nanometer regime with the scanning force microscope. Scanning Microsc 6 (4): 919-29; discussion 929-30, 1992.
Humphries JD, Byron A, Humphries MJ. Integrin ligands at a glance. J Cell Sci 119 (Pt 19): 3901-3, 2006.
Hutter JL, Bechhoefer J. Calibration of Atomic-Force Microscope Tips. Review of Scientific Instruments 64 (7): 1868-1873, 1993.
Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 110 (6): 673-87, 2002.
Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69 (1): 11-25, 1992.
Ingber DE. Fibronectin controls capillary endothelial cell growth by modulating cell shape. Proc Natl Acad Sci U S A 87 (9): 3579-83, 1990.
Khismatullin DB, Truskey GA. A 3D numerical study of the effect of channel height on leukocyte deformation and adhesion in parallel-plate flow chambers. Microvasc Res 68 (3): 188-202, 2004.
Le Grimellec C, Lesniewska E, Cachia C, Schreiber JP, de Fornel F, Goudonnet JP. Imaging of the membrane surface of MDCK cells by atomic force microscopy. Biophys J 67 (1): 36-41, 1994.
Le Grimellec C, Lesniewska E, Giocondi MC, Finot E, Vie V, Goudonnet JP. Imaging of the surface of living cells by low-force contact-mode atomic force microscopy. Biophys J 75 (2): 695-703, 1998.
Li F, Redick SD, Erickson HP, Moy VT. Force measurements of the alpha5beta1 integrin-fibronectin interaction. Biophys J 84 (2 Pt 1): 1252-62, 2003.
Lo CM, Wang HB, Dembo M, Wang YL. Cell movement is guided by the rigidity of the substrate. Biophys J 79 (1): 144-52, 2000.
Lugmaier RA, Hugel T, Benoit M, Gaub HE. Phase contrast and DIC illumination for AFM hybrids. Ultramicroscopy 104 (3-4): 255-60, 2005.
Luo BH, Carman CV, Springer TA. Structural basis of integrin regulation and signaling. Annu Rev Immunol 25: 619-47, 2007.
Luo BH, Springer TA. Integrin structures and conformational signaling. Curr Opin Cell Biol 18 (5): 579-86, 2006.
Massia SP, Hubbell JA. Immobilized amines and basic amino acids as mimetic heparin-binding domains for cell surface proteoglycan-mediated adhesion. J Biol Chem 267 (14): 10133-41, 1992.
McClay DR, Wessel GM, Marchase RB. Intercellular recognition: quantitation of initial binding events. Proc Natl Acad Sci U S A 78 (8): 4975-9, 1981.
Puech PH, Poole K, Knebel D, Muller DJ. A new technical approach to quantify cell-cell adhesion forces by AFM. Ultramicroscopy 106 (8-9): 637-44, 2006.
Puech PH, Taubenberger A, Ulrich F, Krieg M, Muller DJ, Heisenberg CP. Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy. J Cell Sci. Vol. 118, pp. 4199-206, 2005.
Putman CA, van der Werf KO, de Grooth BG, van Hulst NF, Greve J. Viscoelasticity of living cells allows high resolution imaging by tapping mode atomic force microscopy. Biophys J 67 (4): 1749-53, 1994.
Radmacher M. Studying the mechanics of cellular processes by atomic force microscopy. Methods Cell Biol 83: 347-72, 2007.
Radmacher M, Fritz M, Hansma PK. Imaging soft samples with the atomic force microscope: gelatin in water and propanol. Biophys J 69 (1): 264-70, 1995.
Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science 238 (4826): 491-7, 1987.
Salchert K, Oswald J, Streller U, Grimmer M, Herold N, Werner C. Fibrillar collagen assembled in the presence of glycosaminoglycans to constitute bioartificial stem cell niches in vitro. J Mater Sci Mater Med 16 (6): 581-5, 2005.
Sheng X, Ting YP, Pehkonen SO. Force measurements of bacterial adhesion on metals using a cell probe atomic force microscope. J Colloid Interface Sci 310 (2): 661-9, 2007.
Solon J, Levental I, Sengupta K, Georges PC, Janmey PA. Fibroblast adaptation and stiffness matching to soft elastic substrates. Biophys J 93 (12): 4453-61, 2007.
Stossel TP. On the crawling of animal cells. Science 260 (5111): 1086-94, 1993.
Taubenberger A, Cisneros DA, Friedrichs J, Puech PH, Muller DJ, Franz CM. Revealing early steps of alpha2beta1 integrin-mediated adhesion to collagen type I by using single-cell force spectroscopy. Mol Biol Cell 18 (5): 1634-44, 2007.
Thamilselvan V, Basson MD. Pressure activates colon cancer cell adhesion by inside-out focal adhesion complex and actin cytoskeletal signaling. Gastroenterology 126 (1): 8-18, 2004.
Thie M, Rospel R, Dettmann W, Benoit M, Ludwig M, Gaub HE, Denker HW. Interactions between trophoblast and uterine epithelium: monitoring of adhesive forces. Hum Reprod 13 (11): 3211-9, 1998.
Thoumine O, Kocian P, Kottelat A, Meister JJ. Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis. Eur Biophys J 29 (6): 398-408, 2000.
Thoumine O, Ott A, Louvard D. Critical centrifugal forces induce adhesion rupture or structural reorganization in cultured cells. Cell Motil Cytoskeleton 33 (4): 276-87, 1996.
Ting-Beall HP, Needham D, Hochmuth RM. Volume and osmotic properties of human neutrophils. Blood 81 (10): 2774-80, 1993.
Vakonakis I, Campbell ID. Extracellular matrix: from atomic resolution to ultrastructure. Curr Opin Cell Biol 19 (5): 578-83, 2007.
Vesenka J, Mosher C, Schaus S, Ambrosio L, Henderson E. Combining optical and atomic force microscopy for life sciences research. Biotechniques 19 (2): 240-8, 849, 852-3, 1995.
Walker JL, Fournier AK, Assoian RK. Regulation of growth factor signaling and cell cycle progression by cell adhesion and adhesion-dependent changes in cellular tension. Cytokine Growth Factor Rev 16 (4-5): 395-405, 2005.
Wang CC, Hsu YC, Su FC, Lu SC, Lee TM. Effects of passivation treatments on titanium alloy with nanometric scale roughness and induced changes in fibroblast initial adhesion evaluated by a cytodetacher. J Biomed Mater Res A, 2008.
Wojcikiewicz EP, Abdulreda MH, Zhang X, Moy VT. Force spectroscopy of LFA-1 and its ligands, ICAM-1 and ICAM-2. Biomacromolecules 7 (11): 3188-95, 2006.
Wojcikiewicz EP, Zhang X, Chen A, Moy VT. Contributions of molecular binding events and cellular compliance to the modulation of leukocyte adhesion. J Cell Sci 116 (Pt 12): 2531-9, 2003.
Wojcikiewicz EP, Zhang X, Moy VT. Force and Compliance Measurements on Living Cells Using Atomic Force Microscopy (AFM). Biol Proced Online 6: 1-9, 2004.
Yeung T, Georges PC, Flanagan LA, Marg B, Ortiz M, Funaki M, Zahir N, Ming W, Weaver V, Janmey PA. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil Cytoskeleton 60 (1): 24-34, 2005.
Zaari N, Rajagopalan P, Kim SK, Engler AJ, Wong JY. Photopolymerization in microfluidic gradient generators: Microscale control of substrate compliance to manipulate cell response. Advanced Materials 16 (23-24): 2133-+, 2004.
Zhang X, Chen A, De Leon D, Li H, Noiri E, Moy VT, Goligorsky MS. Atomic force microscopy measurement of leukocyte-endothelial interaction. Am J Physiol Heart Circ Physiol 286 (1): H359-67, 2004.
Zhang X, Wojcikiewicz E, Moy VT. Force spectroscopy of the leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction. Biophys J 83 (4): 2270-9, 2002.
Zhong S, Li H, Chen XY, Cao EH, Jin G, Hu KS. Different interactions between the two sides of purple membrane with atomic force microscope tip. Langmuir 23 (8): 4486-93, 2007.
Zhu C. Kinetics and mechanics of cell adhesion. J Biomech 33 (1): 23-33, 2000.
許韶容. 基材軟硬度對上皮細胞及纖維母細胞之黏著力的影響,
2007.