跳到主要內容

簡易檢索 / 詳目顯示

研究生: 邢永年
Yong-Nian Xing
論文名稱: 結合基板引導膠原蛋白自組裝法與利用紫外光曝光及胰蛋白酶蝕刻產生微米圖案的技術來製作膠原蛋白纖維島嶼以應用於細胞表現型之控制
Control of cell phenotype by collagen fibril islands fabricated by substrate-guided self-assembly of collagen molecules and micro-patterning using masked UV exposure and trypsin etching
指導教授: 陳賜原
Szu-yuan chen
口試委員:
學位類別: 碩士
Master
系所名稱: 理學院 - 物理學系
Department of Physics
論文出版年: 2019
畢業學年度: 107
語文別: 中文
論文頁數: 45
中文關鍵詞: 膠原蛋白微米圖案幹細胞原子力顯微鏡
外文關鍵詞: Collagen, Micro-patterning, MSC, AFM
相關次數: 點閱:16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
  • 研究顯示,微米及奈米尺度的基板會影響貼附在上面的細胞,包括貼附形狀、遷移、繁殖、分化等等。細胞外基質像膠原蛋白纖維這種具有特殊結構的基板在控制細胞的表現型上面就扮演了重要的角色。在本實驗中,我們結合自組裝膠原蛋白分子和微米圖案光刻製造膠原蛋白纖維島嶼,我們把人類間充質幹細胞(MSC)養在膠原蛋白纖維島嶼基板上面,觀察細胞是否會貼附在島嶼上面並改變自身形狀,並在基板製作時滴入PLL-g-PEG這種聚合物,之後觀察細胞貼附在島嶼上的情形分析PLL-g-PEG對基板的影響,藉由觀察細胞貼附的狀況來改善PLL-g-PEG的參數,最終達到完全控制細胞的貼附形狀。未來,我們會加入不同的分化劑來觀察MSC貼附在不同的膠原蛋白纖維島嶼上的分化速度,再用共厄焦顯微鏡來觀察細胞內的變化,像是用DNA、F-atin等等,利用觀察的結果改變膠原蛋白纖維島嶼的形狀,讓MSC分化的速度到達最快。


    It has been known that the microscale and nanoscale substrate topography of cell-culturing substrate can regulate cell adhesion, migration, proliferation, and differentiation. It is also known that extracellular matrix is made of most importantly collagen fibrils, which possess specific molecular identity and structural characteristics that may be crucial for controlling cell behaviors such as cell phenotype. In this work, we combined the techniques of self-assembly of collagen molecules and lithographic micropatterning to produce collagen fibril islands. The collagen fibril island substrates were used to culture human mesenchymal stem cells (MSC) to control their shape. We also added PLL-g-PEG on the substrate and observed the cell type to know PLL-g-PEG contribution. In future, We will observe MSC differentiation rate by various collagen fibril islands.We will use DNA, F-actin to observe cell internal transform by collagen fibril islands.

    Abstract IV 摘要 V 致謝 VI 目錄 VII 圖目錄 IX 一.緒論 1 1.1表觀遺傳學 1 1.2細胞外基質 1 1.3膠原蛋白纖維 1 1.4控制細胞貼附大小影響細胞分化 5 1.5 PLL-g-PEG鈍化基板表面 10 1.6 膠原蛋白的光分解 11 二.實驗材料與方法 13 2.1 膠原蛋白薄膜製備 13 2.2 膠原蛋白纖維島嶼製程 14 2.3 強化細胞對膠原蛋白島嶼的貼附性 15 2.4 間充質幹細胞的培養 16 2.5 光蝕刻裝置檢測 16 2.6 膠原蛋白微米圖案檢測 18 2.7 膠原蛋白螢光染色 19 2.8 DAPI與Phalloidin螢光染色 19 2.9 引導MSC分化成脂肪細胞 19 2.10 Oil red染色流程 20 三.結果 21 3.1曝光架設的改進 21 3.2 膠原蛋白薄膜與島嶼 21 3.3膠原蛋白的熱處理(本實驗由Saitong Muneekaew 提供數據) 23 3.4 PLL-g-PEG對膠原蛋白纖維島嶼的影響 24 3.5 MSC在不同PLL-g-PEG時間下的影響 25 3.6引導MSC分化成脂肪細胞 29 四.結論與未來展望 31 4.1 結論 31 4.2未來展望 31 五.參考文獻 33

    [1] F. Guilak, D. M. Cohen, B. T. Estes, J. M. Gimble, W. Liedtke, C. S. Chen, Control of Stem Cell Fate by Physical Interactions with the Extracellular Matrix, CELL STEM CELL 5 (1) (2009) 17–26. doi:{10.1016/j.stem.2009.06.016}.

    [2] M. J. Dalby, N. Gadegaard, R. Tare, A. Andar, M. O. Riehle, P. Herzyk, C. D. W. Wilkinson, R. O. C. Oreffo, The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder,NATUREMATERIALS6(12)(2007)997– 1003. doi:{10.1038/nmat2013}.

    [3] Biomimetic nanopatterns as enabling tools for analysis and control of live cells.Deok-Ho Kim, Hyojin Lee, Young Kwang Lee, Jwa-Min Nam, Andre Levchenko Adv Mater. 2010 Nov 2; 22(41): 4551–4566. doi: 10.1002/adma.201000468

    [4] M. R. Lee, N. Prasain, Y.-J. Kim, M. C. Yoder, H. E. Broxmeyer, Human Embryonic Stem Cell-Specific Micrornas Promote Full Programming of Induced Pluripotent Stem Cells Derived From CD34+Cord Blood Cells Stored Frozen for More Than 20 Years, BLOOD 116 (21) (2010) 1083, 52nd Annual Meeting of the American-Society-of-Hematology (ASH), Orlando, FL, DEC 04-07, 2010.

    [5] F. Pan, M. Zhang, G. Wu, Y. Lai, B. Greber, H. R. Schoeler, L. Chi, Topographic effect on human induced pluripotent stem cells differentiation towards neuronal lineage, BIOMATERIALS 34 (33) (2013) 8131–8139. doi:{10.1016/j. biomaterials.2013.07.025}.

    [6] Collagens--structure, function, and biosynthesis.K. Gelse, E. Pöschl, T. Aigner Adv Drug Deliv Rev. 2003 Nov 28; 55(12): 1531–1546.

    [7] Molecular-scale topographic cues induce the orientation and directional movement of fibroblasts on two-dimensional collagen surfaces. Kate Poole, Khaled Khairy, Jens Friedrichs, Clemens Franz, David A. Cisneros, Jonathon Howard, Daniel Mueller J Mol Biol. 2005 Jun 3; 349(2): 380–386. Published online 2005 Apr 9. doi: 10.1016/j.jmb.2005.03.06

    [8] A. V. Taubenberger, M. A. Woodruff, H. Bai, D. J. Muller, D. W. Hutmacher, The effect of unlocking RGD-motifs in collagen I on pre-osteoblast adhesion and differentiation, BIOMATERIALS 31 (10) (2010) 2827–2835. doi:{10.1016/j. biomaterials.2009.12.051}.

    [9] Wee Wen Leow and Wonmuk Hwang, “Epitaxially Guided Assembly of Collagen Layers on Mica Surfaces”, Langmuir, Vol.27, pp10907–10913, July 2011

    [10] Fengzhi Jiang, Heinrich Hörber, Jonathon Howard and Daniel J. Müller, “Assembly of collagen into microribbons: effects of pH and electrolytes”, Journal of Structural Biology, Vol.148, Issue 3, pp268-278, December 2004

    [11] Molecular-scale topographic cues induce the orientation and directional movement of fibroblasts on two-dimensional collagen surfaces.Kate Poole, Khaled Khairy, Jens Friedrichs, Clemens Franz, David A. Cisneros, Jonathon Howard, Daniel Mueller J Mol Biol. 2005 Jun 3; 349(2): 380–386. Published online 2005 Apr 9. doi: 10.1016/j.jmb.2005.03.064

    [12] WenLi,BofanZhu, ZuzanaStrakova, RongWang, “Two-way regulation between cells and aligned collagen fibrils: Local 3D matrix formation and accelerated neural differentiation of human decidua parietalis placental stem cells” Elsevier June 2014

    [12] Design of Chemically Activated Polymer Microwells by One-Step UV-Lithography for Stem Cell Adhesion Sabrina Montero-Pancera, Vanessa Trouillet, Andre Petershans, Dagmar Fichtner, Andrey Lyapin, Michael Bruns, Thomas Schimmel, Doris Wedlich, Stefan Reichlmaier, Peter G. Weidler, and Hartmut Gliemann Langmuir 2010 26 (3), 2050-2056 DOI: 10.1021/la902563d

    [13] Farshid Guilak, Daniel M. Cohen, Bradley T. Estes, Jeffrey M. Gimble, Wolfgang Liedtke and Christopher S. Chen, “Control of Stem Cell Fate by Physical Interactions with the Extracellular Matrix”, Cell Stem Cell, Vol.5, Issue 1, pp17-26, July 2009.

    [14 ] Patterning of 293T fibroblasts on a mica surface.Sen Hou, Xin-Xin Li, Xiao-Yu Li, Xi-Zeng Feng, Li Guan, Yan-Lian Yang, Chen Wang Anal Bioanal Chem. 2009 Aug; 394(8): 2111–2117. Published online 2009 Jun 25. doi: 10.1007/s00216-009-2892-8

    [15] Nanogrooved substrate promotes direct lineage reprogramming of fibroblasts to functional induced dopaminergic neurons.Junsang Yoo, Myungkyung Noh, Hongnam Kim, Noo Li Jeon, Byung-Soo Kim, Jongpil KimBiomaterials. 2015 Mar; 45: 36–45. Published online 2015 Jan 13. doi: 10.1016/j.biomaterials.2014.12.049

    [16] Boxshall, K. , Wu, M. , Cui, Z. , Cui, Z. , Watts, J. F. and Baker, M. A. (2006), Simple surface treatments to modify protein adsorption and cell attachment properties within a poly(dimethylsiloxane) micro‐bioreactor. Surf. Interface Anal., 38: 198-201. doi:10.1002/sia.2274

    [17] Wei Song, Naoki Kawazoe, and Guoping Chen, “Dependence of Spreading and Differentiation of Mesenchymal Stem Cells on Micropatterned Surface Area,” Journal of Nanomaterials, vol. 2011, Article ID 265251, 9 pages, 2011

    [18] Biophysical regulation of epigenetic state and cell reprogramming.Timothy L. Downing, Jennifer Soto, Constant Morez, Timothee Houssin, Ashley Fritz, Falei Yuan, Julia Chu, Shyam Patel, David V. Schaffer, Song Li Nat Mater. 2013 Dec; 12(12): 1154–1162. Published online 2013 Oct 20. doi: 10.1038/nmat3777

    [19] Microcontact printing of novel co-polymers in combination with proteins for cell-biological applications. Gabor Csucs, Roger Michel, Jost W. Lussi, Marcus Textor, Gaudenz Danuser Biomaterials. 2003 May; 24(10): 1713–1720

    [20] Poly(l-lysine)-g-Poly(ethylene glycol) Layers on Metal Oxide Surfaces:  Attachment Mechanism and Effects of Polymer Architecture on Resistance to Protein Adsorption
    Gregory L. Kenausis,‡, Janos Vörös,‡, Donald L. Elbert,§, Ningping Huang,‡, Rolf Hofer,‡, Laurence Ruiz-Taylor,‡,‖, Marcus Textor,‡, Jeffrey A. Hubbell,§ and, and Nicholas D. Spencer*,‡The Journal of Physical Chemistry B 2000 104 (14), 3298-3309 DOI: 10.1021/jp993359m

    [21] Jariashvili K, Madhan B, Brodsky B, Kuchava A, Namicheishvili L, Metreveli N. UV damage of collagen: insights from model collagen peptides. Biopolymers. 2011;97(3):189-98.

    QR CODE
    :::