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研究生:張家誠
研究生(外文):Chia-Cheng Chang
論文名稱:利用雙光子螢光及二倍頻顯微術觀測外來作用力對於由幹細胞分化的軟骨母細胞分泌膠原蛋白的影響
論文名稱(外文):Utilizing two-photon fluorescence and second harmonic generation microscopy to study the effect of force in stem cell based chondrogensis
指導教授:董成淵
指導教授(外文):Chen-Yan Dong
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:物理研究所
學門:自然科學學門
學類:物理學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:45
中文關鍵詞:間葉幹細胞軟骨母細胞膠原蛋白雙光子顯微術二倍頻
外文關鍵詞:MSCschondrocytecollagentwo-photon microscopysecond harmonic generation microscopy
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軟骨的損傷一直是人體一項不可避免的疾病。而且對於運動員而言,長期持續的運動使的軟骨更容易受傷。一開始的作法是從身體其他部分取出不會影響到正常生理機能的軟骨去填補受傷的部份。不過漸漸的,所需要的軟骨組織越來越多,於是開始試著製作軟骨,這就是組織工程中一個很大的題目。在眾多細胞來源中,利用幹細胞配合適當的鷹架跟生長因子來培養軟骨組織一直受到很大的重視,而怎樣適當的參數會影響到軟骨分泌膠原蛋白也廣被研究。

本實驗即是利用雙光子及倍頻顯微術,觀察受力 ( 0G, 10000G, 20000G,每天施力15分鐘)的軟骨母細胞,其膠原蛋白生長的型態和情形。(一共培養四個禮拜。) 我們觀察到有施力的細胞,膠原蛋白有成束狀的發展,且有局部生長的情形發生。
Rheumatoid arthritis (RA) is a major disease for human. For a sportsman, longtime motion makes articular cartilage hurt easily. Initially we take some cartilage from other part in body to repair the hurt part. Because the more cartilage needed, we try to make the cartilage from cells. This is a big term in tissue engineering. Between all the cell’s sources, we pay much attention to use the stem cells with proper scaffolds and transfer growth factors to cultivate cartilage.

In my experiment, I utilize the auto-fluorescence and second harmonic generation to observe the forced chondrocytes producing collagen.We cultivate mesenchymal stem cells (MSCs) in a medium with TGF-β3 added. TGF-β3 induces the MSCs to differentiate into chondrocytes and the chondrocytes subsequently produce collagen. During culture, we impart force on the cells by using a centrifuge. We choose three force-0G,10000G,200000G and exerted the force on the cells for 15 minutes a day for 4 weeks. We observed that the cells exerted with greater force produce collagen that tend to accumulate in lumps at early stage, and generate stronger second harmonic signal.
目錄
致謝
目錄
圖目錄
摘要
Abstract
第一章 緒論
1-1 前言 9
9
1-2 組織工程 9
1.2.1 細胞 10
1.2.2 鷹架 11
1.2.3 訊息因子 11
1-3 軟骨 12
1-4 膠原蛋白 13
第二章 實驗原理
2-1 雙光子顯微術 14
14
2-2 二倍頻 22
第三章 儀器架設
3-1 雷射系統 24
24
3-2 光路元件 24
3-3 顯微系統 24
3-4 訊號接收 25
3-5 掃描系統 25
第四章 實驗步驟與方法
4-1 樣品來源 27
27
4-2 培養液 27
4-3 觀測裝置 27
4-4 樣品處理 28
4-5 顯微系統 28
第五章 實驗結果與分析 30

第六章 結論 43

第七章 參考文獻 44
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2.楊婷琪 “組織工程相關產品及重要公司分析” (民91)
3.Gomes ME, Reis RL. “Tissue engineering: Key Elements and Some Trends.” Macromolecular Bioscience 4 (8): 737-742 AUG 9 2004
4.Maeda S, Nishida J, Sato T, Inomata Y, Shimamura T, Horiuchi S. “Changes in microstructure and gene expression of articular chondrocytes cultured in a tube under mechanical stress.” OSTEOARTHRITIS AND CARTILAGE 13 (2): 154-161 FEB 2005
5.LeBaron RG, Athanasiou KA. “Ex vivo synthesis of articular cartilage.” BIOMATERIALS 21 (24): 2575-2587 Sp. Iss. SI, DEC 2000
6.Ross/Romrell著 林志聰等人譯 “組織學” (1989)
7.Hsuan-Shu Lee, Shu-Wen Teng.” Imaging Human Bone Marrow Stem Cell Morphogenesis in Polyglycolic Acid Scaffold by Multiphoton Microscopy.”Tissue Engineering 12 (10) 2006
8.Peter T. C. So, Chen Y. Dong “Two-Photon Excitation Fluorescence Microscopy.” Bio-methods 02, 999-429, 2000
9.R.G. LeBaron, K.A. Athanasiou. “Ex Vivo synthesis of articular cartilage.” Biomaterials 21, 2575-2587, 2000
10.Adrian C. Shieh, Kyriacos A. Athanasiou “Pinciples of Cell Mechanics for Cartilage Tissue Engineering.” Biomedical Engineering Society (31) ,1-11 ,2003
11.Aivin T. Yeh, Marie J. Hammer-Wilson. “Nonlinear optical microscopy of articular cartilage” Osteoarthritis and Cartilage 13, 345-352,2005
12.Johnna S. Temenoff, Antonios G. Mikos. “Review: tissue engineering for regeneration of articular cartilage.” Biomaterials 21, 431-440, 2000
13.Stock, U.A., and Vacanti, J. P. “Tissue engineering: current state and prospects.” Annu. Rev. Med. 52, 443, 2001
14.Freed, L.E., and Vuhjak-Novaskovic, G. “tissue culture bioreactors: chondrogenesis as a model system.” In: Lanza R.P., Langer, R., and Chick, W.L., eds. Principles of Tissue Engineering. San Diego: Academic Priss, 1997,pp.155-165.
15.Caplan, A.I., and Bruder, S.P. “Mesenchymal stem cells:building blocks for molecular medicine in the 21st sentruy.”Trends Mol. Med. 7, 259, 2001
16.Caterson, E.J., Nesti, L.J., Li, W.J., Danielson, K.G., Albert, T.J., Vaccaro, A.R., et al. “Three-dimensional cartilage for mation by bone marrow-derived cells seeded in polylactide / alginate amalgam.” J. Biomed. Mater. Res. 57, 394, 2001
17.Lee, G.S., Huang, G.T., Chiang, H., Chiou, L.L., Chen, M.H.,Hsieh C.H., et al. “Multipotential mesenchymal stem cells from femoral bone marrow near the site of osteonecrosis.” Stem Cell 21, 190, 2003.
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