現在位置首頁 > 博碩士論文 > 詳目
論文中文名稱:比較動態拉伸頻率對包覆於褐藻酸鈉水膠纖維中纖維母細胞生長與型態的影響 [以論文名稱查詢館藏系統]
論文英文名稱:Effects of cyclic stretching on the growth and morphology of fibroblasts embedded in alginate hydrogel fibers [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:化學工程研究所
畢業學年度:102
出版年度:103
中文姓名:吳旻哲
英文姓名:Min-Che Wu
研究生學號:101738012
學位類別:碩士
語文別:中文
口試日期:2014-07-01
論文頁數:51
指導教授中文名:林忻怡
口試委員中文名:黃聲東;王大銘
中文關鍵詞:機械刺激褐藻酸鈉纖維母細胞快速成型
英文關鍵詞:Mechanical stimulusAlginateFibroblastRapid prototyping
論文中文摘要:之前的研究發現,若將細胞包覆在褐藻酸鈉水膠之中,生長於褐藻酸鈉水膠纖維中的纖維母細胞,能夠有較快的細胞生長和較佳基因表現,但大多數的拉伸試驗都是將細胞種在材料表面,故本實驗將細胞包覆在5% RGD改質的褐藻酸鈉水膠中,並用快速原型系統製作成3D多孔隙支架,每天以15rpm及60rpm的頻率拉伸樣品1分鐘,而對照組則是靜態培養。於第1、4、7、14天,觀察細胞生長在不同環境下的細胞型態、增生、活性的差異。
在活細胞即時觀察影像系統下發現,實驗組及對照組的細胞會在纖維中移動。DNA定量結果顯示,第7天後實驗組的細胞比對照組生長的還要快。在早期(第1天及第4天)對照組的細胞活性明顯高於實驗組。隨著培養時間的增加(第7天及第14天),實驗組細胞的活性增加並超過了對照組的細胞。Live/Dead染色發現,細胞在1,4,7,14天大多是存活的。HE染色顯示纖維母細胞在拉伸後的14天內沒有明顯的細胞型態改變,但分散均勻,很少團聚。在第一型膠原蛋白免疫螢光染色觀察下,發現膠原蛋白圍繞著細胞,膠原蛋白也無增加的趨勢。
論文英文摘要:In this experiment, cell was mixed in 5% RGD-alginate and used rapid prototyping system to build the 3D porous scaffolds. The experimental group (E) was cyclically stretched for 1 min per day with a frequency of 15 rpm (E-15) and 60 rpm (E-60), respectively; the control group (C) was static culture. The cells were harvested on day 1, 4, 7, 14.
The cell proliferation results show that the cells embedded in alginate fibers grew overtime and that day 7 the cells subjected to stretching grew slightly faster compared to those under static culture. The viability of cells under static culture was higher than that of stretched cells in earlier days (day 1 and 4). As the culture time increased (day 7 and 14), the viability of stretched cells increased and surpassed that of static cells. We found that live/dead staining appeared the cells were mostly survival in day 1, 4, 7, 14. HE staining showed fibroblast stained cultures after 14 days of cyclic stretch have no apparent preferential orientation. Others have shown stretching of cells changes cell morphology. We did not observe this in our experiments. Immunofluorescence staining showed that type I collagen found around the cell and no collagen increased.
論文目次:摘 要 i
ABSTRACT ii
誌 謝 iii
目 錄 iv
表目錄 vi
圖目錄 vii
第一章 緒論 1
1.1 前言 1
1.2 研究目的 2
第二章 文獻回顧 3
2.1 傷口癒合過程 3
2.2 快速原型系統 5
2.3 機械應力對細胞的影響 6
2.4 褐藻酸鈉 9
2.5 氨基酸序列分子 11
第三章 實驗材料與方法 12
3.1 實驗材料 12
3.1.1 細胞來源 12
3.1.2 細胞培養用藥品 12
3.1.3 實驗藥品 13
3.1.4 儀器設備 15
3.1.5 藥品及溶液製備 17
3.2 實驗方法 20
3.2.1 實驗設計 20
3.2.2 RGD-褐藻酸鈉濃度、拉伸參數之選擇 20
3.2.3 實驗流程 21
3.2.4 立體繪圖系統架設 22
3.2.5 動態拉伸 24
3.2.6 製作RGD改質之褐藻酸鈉 25
3.2.7 製作RGD改質之褐藻酸鈉-細胞支架 26
3.2.8 纖維母細胞在支架上的活性測試 27
3.2.9 統計分析 30
第四章 結果與討論 31
4.1 細胞型態與分布 31
4.1.1 活細胞即時觀察影像系統 31
4.1.2 細胞存活螢光染色 32
4.1.3 Haematoxylin & Eosin染色 35
4.2 細胞增生 40
4.3 細胞活性測定 41
4.4 第一型膠原蛋白免疫螢光染色 42
第五章 結論 44
參考文獻 45
附錄 50
論文參考文獻:1.Miron-Mendoza M, Koppaka V, Zhou C, Petroll WM. Techniques for assessing 3-D cell–matrix mechanical interactions in vitro and in vivo. Experimental cell research 319: 2470–2480, 2013.
2.Mcgarry James G., Klein-Nulend Jenneke, Prendergast Patrick J. The effect of cytoskeletal disruption on pulsatile fluid flow-induced nitric oxide and prostaglandin E2 release in osteocytes and osteoblasts. Biochemical and Biophysical Research Communications 330 (1): 341-348, 2005.
3.Thi MM., Kojima T., Cowin SC., et al. C. Fluid shear stress remodels expression and function of junctional proteins in cultured bone cells. Am J Physiol Cell Physiol 284 (2): 389-403, . 2003.
4.Norvell SM., Ponik SM., Bowen DK., et al. Fluid shear stress induction of COX-2 protein and prostaglandin release in cultured MC3T3-E1 osteoblasts does not require intact microfilaments or microtubules. J Appl Physiol 96 (3): 957-966, 2004.
5.Ponik SM., Pavalko FM. Formation of focal adhesions on fibronectin promotes fluid shear stress induction ofCOX-2 and PGE2 release inMC3T3-E1 osteoblasts. JAppl Physiol 97 (1): 135-142, 2004.
6.Morita Tsuyoshi, Mayanagi Taira, Sobue Kenji. Reorganization of the actin cytoskeleton via transcriptional regulation of cytoskeletal/focal adhesion genes by myocardin-related transcription factors (MRTFs/MAL/MKLs). Experimental Cell Research 313 (16): 3432-3445, 2007.
7.Launay Nathalie, Goudeau Bertrand, Kato Kanefusa,et al. Cell signaling pathways to [alpha]B-crystallin following stresses of the cytoskeleton. Experimental Cell Research 312 (18): 3570-3584, 2006.
8.Lee DY., Yeh CR., Chang SF.,et al. Integrin-mediated expression of bone formation-related genes in osteoblastlike cells in response to fluid shear stress: roles of extracellular matrix, Shc, and mitogen-activated protein kinase. J BoneMiner Res 23 (7): 1140-1149, 2008.
9.Mccormick SM., Saini V., Yazicioglu Y., et al. Interdependence of pulsed ultrasound and shear stress effects on cell morphology and gene expression. Ann Biomed Eng 34 (3): 436-445, 2006.
10.Jon A. Rowley, Gerard Madlambayan, David J. Mooney. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20: 45-53, 1999.
11.Wei-Wen Wu. Cell Embedded in Fibers of Porous Scaffold through 3D Plotting Fabrication to Repair Skin. National Taipei University of Technology;2012.
12.I.K. Cohen, et al. An update on wound healing. Ann Plast Surg 3 (3): 264-272, 1979.
13.D. G. Greenhalgh. The role of apotosis in wound healing. Int J Biochem Cell Biol 30 (9): 1019-1030, 1998.
14.D. M. Simpson and R. Ross. The neutrophilic leukocyte in wound repair a study with antineutrophil serum. J Clin Invest 51 (8): 2009-2023, 1972.
15.P. Martin. Wound healing—aiming for perfect skin regeneration. Science 276 (5309): 75-81, 1997.
16.Sandra A L Moura, Luiza Dias C Lima, Sílvia Passos Andrade, Armando Da Silva-Cunha, Rodrigo L Órefice, Eliane Ayres, Gisele Rodrigues Da Silva. The role of macrophages in wound repair: a review. Plast Reconstr Surg 68 (1): 107-113, 1981.
17.Behrooz A. Torkian, MD; Alvin T. Yeh, PhD; Rodney Engel, BA; Chung-Ho Sun, PhD; Bruce J. Tromberg, PhD; Brian J. F. Wong, MD, PhD. Modeling aberrant wound healing using tissue-engineered skin constructs and multiphoton microscopy. Arch Facial Plast Surg 6 (3): 180-187, 2004.
18.http://www.dr-huang.com.tw/huangword03.asp
19.Ma, P.X., Scaffolds for tissue fabrication. Materials Today 7 (5): 30-40, 2004.
20.Almirall A, Larrecq G, Delgado J.A., Martínez S., Planell J.A., Ginebra M.P. Fabrication of low temperature macroporous hydroxyapatite scaffolds by foaming and hydrolysis of an α-TCP paste. Biomaterials 25 (17): 3671-3680, 2004.
21.Leong, K.F., C.M. Cheah, and C.K. Chua. Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs. Biomaterials 24 (13): 2363-2378, 2003.
22.Zein I, Hutmacher D.W., Tan K.C., Teoh S.H. Fused deposition modeling of novel scaffold architectures for tissue engineering applications. Biomaterials 23 (4): 1169-1185, 2002.
23.Tan K.H., Chua C.K., Leong K.F., Cheah C.M., Cheang P., Abu Bakar M.S., Cha S.W. Scaffold development using selective laser sintering of polyetheretherketone-hydroxyapatite biocomposite blends. Biomaterials 24 (18): 3115-3123, 2003.
24.S.S. Kim, H. Utsunomiya, J.A. Koski, B.M. Wu, M.J. Cima, J. Sohn, K. Mukai, L.G. Griffith, J.P. Vacanti. Survival and function of hepatocytes on a novel three-dimensional synthetic biodegradable polymer scaffold with an intrinsic network of channels. Ann Surg 228 (1): 8-13, 1998.
25.Alejandro Nieponice, Timothy M. Maul, Joy M. Cumer, Lorenzo Soletti, David A. Vorp. Mechanical stimulation induces morphological and phenotypic changes in bone marrow-derived progenitor cells within a three-dimensional fibrin matrix. J Biomed Mater Res A. 1; 81(3): 523-30, 2007.
26.Siddarth D.Subramony , AmandaSu , KeithYeager, HelenH.Lu. Combined effects of chemical priming and mechanical stimulation on mesenchymal stem cell differentiation on nanofiber scaffolds. Journal of Biomechanics 47: 2189–2196, 2014
27.Yonggang Pang , Xiaoli Wang , Dongkeun Lee, Howard P. Greisler. Dynamic quantitative visualization of single cell alignment and migration and matrix remodeling in 3-D collagen hydrogels under mechanical force. Biomaterials (32): 3776-3783, 2011.
28.Peter A. Galie, M.S. and Jan P. Stegemann, Ph.D. Simultaneous Application of Interstitial Flow and Cyclic Mechanical Strain to a Three-Dimensional Cell-Seeded Hydrogel. TISSUE ENGINEERING: Part C 17(5): 527-537, 2011.
29.Chenyu Huang, Kunio Miyazaki, Satoshi Akaishi, Atsushi Watanabe, Hiko Hyakusoku, Rei Ogawa. Biological effects of cellular stretch on human dermal fibroblasts. Journal of Plastic, Reconstructive & Aesthetic Surgery 66: e351-e361, 2013.
30.劉錦芬 台灣龍鬚菜萃取物之免疫生理活性探討 國立海洋大學 食品科學研究所 碩士論文(1990)
31.鍾國銘 利用反應曲面法探討幾丁聚醣-褐藻酸鈉複合膜之物化特性與其在中式香腸衣應用 國立海洋大學 食品科學研究所 碩士論文(2003)
32.Fuijiki, K., Yano, T. Effects of sodium alginate on the non-specific defense system of common carp(Cyprinus carpioL.) Journal of Fish Shellfish Imminl. 7: 417-427, 1997
33.林育德 幾丁聚醣與褐藻酸鈉複合水膠之物化特性 國立海洋大學 食品科學研究所 碩士論文(2002)
34.Kimihide Hayakawa, Naruki Sato, and Takashi Obinata. Dynamic Reorientation of Cultured Cells and Stress Fibers under Mechanical Stress from Periodic Stretching. Experimental Cell Research 268: 104–114 , 2001.
35.M. A. Biesalski, A. Knaebel, R. Tu, and M. Tirrell. Cell adhesion on a polymerized pep tide-amphiphile monolayer. Biomaterials (27): 1259-1269, 2006.
36.H. J. Cha, D. S. Hwang, and S. B. Sim. Cell adhesion biomaterial based on mussel adhesive protein fused with RGD peptide. Biomaterials (28): 4039-4046, 2007.
37.Lauffenburger D A, Griffith L G. Proc. Natl. Acad. Sci. U.S.A (98): 4282-4284, 2001.
38.Li JP, de Wijn JR, Van Blitterswijk CA, de Groot K. Porous Ti6Al4V scaffold directly fabricating by rapid prototyping: Preparation and in vitro experiment. Biomaterials (27): 1223-1235, 2006
39.C. Neidlinger-Wilke, E. S. Grood, J. H.-C. Wang, R. A. Brand, L. Claes. Cell alignment is induced by cyclic changes in cell length studies of cells grown in cyclically stretched substrates. Journal of Orthopaedic Research 19: 286-293, 2001.
40.Tali Re’em, Orna Tsur-Gang, Smadar Cohen. The effect of immobilized RGD peptide in macroporous alginate scaffolds on TGFβ1-induced chondrogenesis of human mesenchymal stem cells. Biomaterials (31): 6746-6755, 2010
41.S. Park, S. Lee, and W. Kim. Fabrication of hydrogel scaffolds using rapid prototyping for soft tissue engineering. Macromolecular Research 19 (7): 694-698, 2011.
42.G. Avwioro. Histochemical uses of haematoxylin- A review. J Pharm Clin Sci 1: 24-34, 2011.
43.Yulia Sapir , Smadar Cohen , Gary Friedman , Boris Polyak. The promotion of in vitro vessel-like organization of endothelial cells in magnetically responsive alginate scaffolds. Biomaterials (33): 4100-4109, 2012.
44.Sílvia J. Bidarra, Cristina C. Barrias, Keila B. Fonseca, Mário A. Barbosa, Raquel A. Soares. Pedro L. Granja Injectable in situ crosslinkable RGD-modified alginate matrix for endothelial cells delivery. Biomaterials (32): 7897-7904, 2011.
45.Hunt, N.C., R.M. Shelton, and L. Grover, An alginate hydrogel matrix for the localised delivery of a fibroblast/keratinocyte co-culture. Biotechnol J 4(5): 730-737, 2009.
46.http://www.biology-online.org/dictionary/Anchorage-dependent_cell
47.Potts, J.R. and I.D. Campbell, Structure and function of fibronectin modules. Matrix Biol 15(5): 313-20; discussion 321, 1996.
48.Jeanie L. Drury, Robert G. Dennis, David J. Mooney. The tensile properties of alginate hydrogels. Biomaterials 25: 3187–3199, 2004.
論文全文使用權限:同意授權於2019-08-28起公開