跳到主要內容

臺灣博碩士論文加值系統

(3.235.227.117) 您好!臺灣時間:2021/07/28 03:57
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:施弈安
研究生(外文):YI-AN SHIH
論文名稱:以動態培養探討幾丁聚醣導管中乳牙幹細胞分化成神經細胞的研究
論文名稱(外文):SHED cells differentiate to nerve cells in chitosan conduit under dynamic culture
指導教授:蘇文達蘇文達引用關係
口試委員:柯智升廖永豐陳文章
口試日期:2012-07-20
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:68
中文關鍵詞:乳牙幹細胞動態培養神經分化幾丁聚醣
外文關鍵詞:Stem Cells from Human Exfoliated Deciduous Teethdynamic cultureneural differentiationChitosan
相關次數:
  • 被引用被引用:0
  • 點閱點閱:82
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
乳牙幹細胞(Stem cells from human exfoliated deciduous teeth, SHED)是一種新的幹細胞來源之一。許多研究證實具有類似於間葉幹細胞分化的能力,由於可以直接從脫落的乳牙取得,安全無風險。
本實驗利用凍乾法製作出4%幾丁聚醣神經導管,提供細胞三維空間生長,並搭配旋轉細胞培養系統,藉由動態培養以刺激細胞分化。本研究探討乳牙幹細胞在神經分化上的能力,藉由即時聚合酶鏈式反應分析神經細胞相關基因(Nestin、β-III tubulin、GFAP、CNPase)的表現,並利用共軛焦螢光顯微鏡觀察其培養於材料上之形態與特定神經分化相關蛋白質(Nestin、γ-enolase)的表現。
經細胞培養測試,結果顯示與平面培養之乳牙幹細胞相比,4%幾丁聚醣導管上的細胞能表現大量神經膠細胞特有的GFAP與CNPase基因。神經膠細胞在過去研究都認為是輔助功能居多,近年來越來越多研究的指出它具有協助神經元細胞傳遞電訊號。隨培養時間拉長,神經系統特有的細胞骨架蛋白─β-III tubulin基因表現增加11倍,其被認為是神經元早期的標記。於動態培養下,材料上的乳牙幹細胞形態顯得較聚集;透過支架與動態系統幫助乳牙幹細胞分化為神經細胞,顯示4%幾丁聚醣導管能有效幫助乳牙幹細胞分化成神經膠細胞,而未來可配合動物實驗研究,更進一步可應用在臨床實驗上。


Stem cells from human exfoliated deciduous teeth (SHED) are novel stem cell lines. Many studies have confirmed that SHEDs are similar to mesenchymal stem cell differentiated capacity and can be directly obtained from the dental waste, reducing the complexity of surgery problems.
4% chitosan conduit fabricated by the freeze-dried to provide the cell growth of three-dimensional space. Using a revolving oscillator, promote cell differentiation under dynamic culture system. In this study, we measured neural differentiation capacity of SHEDs by Real-time polymerase chain reaction(qPCR), and detected neuronal differentiation associated gene (Nestin、β-III tubulin、GFAP、CNPase). The morphology of SHED cells on chitosan conduit were observed by using confocal microscopy as well as the immunocytochemistry of Nestin and γ-enolase measured.
Based on the results, the gene expression of glial cell marker GFAP and CNPase under chitosan conduit are higher than the cells those in growing in the plane culture.
In the past, neuroglial cells were an assisting role in neural cells. In recent years, there are many studies indicated that neuroglia can assist the electrical signals. After long time incubation, the cell skeleton protein β-III tubulin of gene expression increased 11 times, β-III tubulin was considered is the early neuronal markers. SHEDs would cluster together in chitosan conduit under dynamic culture. Through the 3D-scaffold and the dynamic system, which can promote SHEDs differentiation into neural cells. Our data indicate that chitosan conduit combined with the dynamic culture significantly help SHEDs differentiation into neuroglial cells. This culture system can be used to animal experimentation, and in the clinical application in future.


目錄

摘 要 i
ABSTRACT iii
誌謝 v
目錄 vi
表目錄 viii
圖目錄 ix
第一章 緒論 1
第二章 文獻回顧 3
2.1組織工程介紹 3
2.2神經系統 5
2.2.1神經元細胞 5
2.2.2神經膠細胞 6
2.3乳牙幹細胞 9
2.3.1乳牙幹細胞介紹 9
2.3.2乳牙幹細胞分離技術 10
2.3.3乳牙幹細胞神經分化潛力 10
2.4神經修復 12
2.4.1組織工程神經導管功能 13
2.4.2神經導管內神經修復過程 14
2.4.3神經導管修復相關研究 15
2.5生物反應器 16
2.6幾丁聚醣 21
2.6.1基本介紹 21
2.6.2幾丁聚醣生醫材料特性 21
第三章 實驗藥品與儀器設備與方法 23
3.1 實驗藥品與儀器設備 23
3.1.1本研究所使用之材料、藥品 23
3.1.2 儀器設備 25
3.2 實驗方法 27
3.2.1實驗架構 27
3.2.2細胞培養 28
3.2.2.1常態培養 28
3.2.2.2 細胞儲存 29
3.2.2.3動態培養 29
3.2.3 細胞培養藥品配製 30
3.2.3.1 PBS配法 30
3.2.3.2細胞培養液配法 30
3.2.3.3 Trypsin-EDTA配製 31
3.2.4幾丁聚醣導管製備 31
3.2.5細胞生長活性測試 32
3.2.6掃瞄式電子顯微鏡觀察 33
3.2.7抽取導管中乳牙幹細胞的total RNA 33
3.2.8 RNA反轉錄cDNA(Reverse transcriptase reaction) 34
3.2.9 Real-Time PCR 35
3.2.10共軛焦顯微鏡-免疫螢光染色 36
3.2.11 流式細胞儀 37
3.2.12幾丁聚醣導管之孔隙度與膨潤度測量 38
第四章 結果與討論 40
4.1材料形態觀察 40
4.1.1材料基本形態 40
4.1.2 4%幾丁聚醣導管膨潤度與孔隙度 40
4.1.3 以電子顯微鏡觀察材料形態 41
4.2細胞形態觀察 44
4.2.1乳牙幹細胞培養於二維環境 44
4.2.2比較不同濃度幾丁聚醣導管培養乳牙幹細胞差異 45
4.2.3 比較乳牙幹細胞分化前後之差異(二維) 46
4.2.4 比較乳牙幹細胞分化前後之差異(三維) 47
4.2.5動態培養 49
4.3乳牙幹細胞生長趨勢 51
4.3.1細胞生長活性測試-二維培養 51
4.3.2細胞生長活性測試-三維培養 52
4.3.3細胞生長活性測試-靜態與動態比較 53
4.3.4乳牙幹細胞培養於幾丁聚醣導管之抗凋亡能力評估 54
4.4乳牙幹細胞神經表現 55
4.4.1流式細胞儀 55
4.4.2 免疫螢光染色(神經幹細胞) 57
4.4.3 免疫螢光染色(神經元細胞) 58
4.4.4 免疫螢光染色(神經膠細胞) 58
4.4.5乳牙幹細胞之神經基因表現 59
第五章 結論 62
參考文獻 64


參考文獻

[1]The National SCI Statistical Center, "Spinal Cord Injury Facts and Figures at a Glance," pp. 1-2, 2011.
[2]D. Dado and S. Levenberg, "Cell-scaffold mechanical interplay within engineered tissue," Semin Cell Dev Biol, vol. 20, pp. 656-664, 2009.
[3]L. V. M. a. A. G. M. Julia E. Babensee, "Growth Factor Delivery for Tissue Engineering," Pharmaceutical Research, vol. 17, pp. 497-504, 2000.
[4]O. D. U. Jean-Pierre Barral, and Alain Croibier, Osteopathe DO MRO (F), "Manual Therapy for the Peripheral Nerves," Elsevier, pp. 11-12, 2007.
[5]H. W. Werner Risau, "Development of the blood-brainbarrier," Trends in Neurosciences, vol. 13, pp. 174-178, 1990.
[6]F. W. Pfrieger, "Synaptic Efficacy Enhanced by Glial Cells in Vitro," Science, vol. 277, pp. 1684-1687, 1997.
[7]E. M. Ullian, "Control of Synapse Number by Glia," Science, vol. 291, pp. 657-661, 2001.
[8]C. F. S. F. H. G. Hongjun Song, "Astroglia induce neurogenesis from adult neural stem cells," Nature, vol. 417, pp. 39-44, 2002.
[9]R. A. S. Christopher M. Anderson, "Astrocyte glutamate transport: Review of properties, regulation, and physiological functions," GILA, vol. 32, pp. 1-14, 2000.
[10]R. D. Fields and B. Stevens-Graham, "New insights into neuron-glia communication," Science, vol. 298, pp. 556-562, 2002.
[11]M. Nedergaard, B. Ransom, and S. A. Goldman, "New roles for astrocytes: Redefining the functional architecture of the brain," Trends in Neurosciences, vol. 26, pp. 523-530, 2003.
[12]E. Gielen, W. Baron, M. Vandeven, P. Steels, D. Hoekstra, and M. Ameloot, "Rafts in oligodendrocytes: evidence and structure-function relationship," Glia, vol. 54, pp. 499-512, 2006.
[13]R. H. Miller and S. Mi, "Dissecting demyelination," Nat Neurosci, vol. 10, pp. 1351-1354, 2007.
[14]I. Griffiths, "Axonal Swellings and Degeneration in Mice Lacking the Major Proteolipid of Myelin," Science, vol. 280, pp. 1610-1613, 1998.
[15]A. S. W. Scott T. Brady, Laura L. Kirkpatrick, Sylvie M. de Waegh, Carol Readhead, Pang-Hsien Tu, and Virginia M.-Y. Lee, "Formation of Compact Myelin Is Required for Maturation of the Axonal Cytoskeleton," The Journal of Neuroscience, vol. 19, pp. 7278-7288, 1999.
[16]H. S. Keirstead, G. Nistor, G. Bernal, M. Totoiu, F. Cloutier, K. Sharp, and O. Steward, "Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury," J Neurosci, vol. 25, pp. 4694-4705, 2005.
[17]R. S. A. F. Huxley, "Evidence for saltatory conduction in peripheral myelinated nerve fibres," The Journal of Physiology, vol. 108, pp. 315-339, 1949.
[18]K. R. Jessen and R. Mirsky, "The origin and development of glial cells in peripheral nerves," Nat Rev Neurosci, vol. 6, pp. 671-682, 2005.
[19]S. M. HALL, "Regeneration in cellular and acellular autografts in the peripheral nervous system," Neuropathology and Applied Neurobiology, vol. 12, pp. 27-46, 1986.
[20]S. G. Songtao Shi, "Perivascular Niche of Postnatal Mesenchymal Stem Cells in Human Bone Marrow and Dental Pulp," Journal of Bone and Mineral Research, vol. 18, pp. 696–704, 2003.
[21]M. Miura, S. Gronthos, M. Zhao, B. Lu, L. W. Fisher, P. G. Robey, and S. Shi, "SHED: stem cells from human exfoliated deciduous teeth," Proc Natl Acad Sci U S A, vol. 100, pp. 5807-5812, 2003.
[22]A. Achilleos and P. A. Trainor, "Neural crest stem cells: discovery, properties and potential for therapy," Cell Res, vol. 22, pp. 288-304, 2012.
[23]J. Wang, X. Wang, Z. Sun, H. Yang, S. Shi, and S. Wang, "Stem cells from human-exfoliated deciduous teeth can differentiate into dopaminergic neuron-like cells," Stem Cells Dev, vol. 19, pp. 1375-1383, 2010.
[24]A. R. Arthur, G.Shi, S.Koblar, S. A.Gronthos, S., "Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues," Stem Cells, vol. 26, pp. 1787-1795, 2008.
[25]G. C. de Ruiter, M. J. Malessy, M. J. Yaszemski, A. J. Windebank, and R. J. Spinner, "Designing ideal conduits for peripheral nerve repair," Neurosurg Focus, vol. 26, p. E5, 2009.
[26]P. T. Clemens van Blitterswijk, Anders Lindahl, Jeffrey Hubbell, David F. Williams, Ranieri Cancedda, Joost D. de Bruijn and Jerome Sohier "Tissue Engineering " Academic Press, vol. 1st Edition, pp. 615-616, 2008.
[27]S. C. J. B. James B. Phillips, Susan M. Hall, and Robert A. Brown., "Neural Tissue Engineering: A Self-Organizing Collagen Guidance Conduit," Tissue Engineering, vol. 11, pp. 1611-1617, 2005.
[28]Y. Yang, W. Zhao, J. He, Y. Zhao, F. Ding, and X. Gu, "Nerve conduits based on immobilization of nerve growth factor onto modified chitosan by using genipin as a crosslinking agent," Eur J Pharm Biopharm, vol. 79, pp. 519-525, 2011.
[29]L. Yao, G. C. de Ruiter, H. Wang, A. M. Knight, R. J. Spinner, M. J. Yaszemski, A. J. Windebank, and A. Pandit, "Controlling dispersion of axonal regeneration using a multichannel collagen nerve conduit," Biomaterials, vol. 31, pp. 5789-5797, 2010.
[30]D. W. Hutmacher and H. Singh, "Computational fluid dynamics for improved bioreactor design and 3D culture," Trends Biotechnol, vol. 26, pp. 166-172, 2008.
[31]P. Sucosky, D. F. Osorio, J. B. Brown, and G. P. Neitzel, "Fluid mechanics of a spinner-flask bioreactor," Biotechnol Bioeng, vol. 85, pp. 34-46, 2004.
[32]S. Saporta, A. E. Willing, R. Shamekh, P. Bickford, D. Paredes, and D. F. Cameron, "Rapid differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor," Brain Res Bull, vol. 64, pp. 347-356, 2004.
[33]M. B. Keogh, S. Partap, J. S. Daly, and F. J. O''Brien, "Three hours of perfusion culture prior to 28 days of static culture, enhances osteogenesis by human cells in a collagen GAG scaffold," Biotechnol Bioeng, vol. 108, pp. 1203-1210, 2011.
[34]D. O. Scott Maxson, and Karen J.L. Burg, "Bioreactors for Tissue Engineering," Tissue Engineering, pp. 179-197, 2011.
[35]C. W. E. H. Sathish Kumar, "Review:Minibioreactors," 26, vol. 26, pp. 1-10, 2004.
[36]W. S. L. Hong-Fang Lu, Peng-Chi Zhang, Ser Mien Chia, Hanry Yu, Hai-Quan Mao, and Kam W. Leong. , "Galactosylated Poly(vinylidene difluoride) Hollow Fiber Bioreactor for Hepatocyte Culture," TISSUE ENGINEERING, vol. 11, pp. 1667-1677, 2006.
[37]R. Portner, S. Nagel-Heyer, C. Goepfert, P. Adamietz, and N. M. Meenen, "Bioreactor design for tissue engineering," J Biosci Bioeng, vol. 100, pp. 235-245, 2005.
[38]H. C. Chen and Y. C. Hu, "Bioreactors for tissue engineering," Biotechnol Lett, vol. 28, pp. 1415-1423, 2006.
[39]C.-J. Wang, F.-S. Wang, K. D. Yang, L.-H. Weng, C.-C. Hsu, C.-S. Huang, and L.-C. Yang, "Shock wave therapy induces neovascularization at the tendon–bone junction. A study in rabbits," Journal of Orthopaedic Research, vol. 21, pp. 984-989, 2003.
[40]L. Yao, L. Shanley, C. McCaig, and M. Zhao, "Small applied electric fields guide migration of hippocampal neurons," J Cell Physiol, vol. 216, pp. 527-535, 2008.
[41]K. P. Ji Sun Park, Hyun Tae Moon, Dae Gyun Woo, Han Na Yang,Keun-Hong Park, "Electrical Pulsed Stimulation of Surfaces Homogeneously Coated with Gold Nanoparticles to Induce Neurite Outgrowth of PC12 Cells," Langmuir, vol. 25, pp. 451-457, 2009.
[42]L. Yao, A. Pandit, S. Yao, and C. D. McCaig, "Electric field-guided neuron migration: a novel approach in neurogenesis," Tissue Eng Part B Rev, vol. 17, pp. 143-153, 2011.
[43]N. Bhattarai, "Nanofibrous Structure of Chitosan for Biomedical Applications," Journal of Nanomedicine & Biotherapeutic Discovery, vol. 02, pp. 1-9, 2012.
[44]J. M. Zuidema, M. M. Pap, D. B. Jaroch, F. A. Morrison, and R. J. Gilbert, "Fabrication and characterization of tunable polysaccharide hydrogel blends for neural repair," Acta Biomater, vol. 7, pp. 1634-1643, 2011.
[45]Y. C. Kuo, C. F. Yeh, and J. T. Yang, "Differentiation of bone marrow stromal cells in poly(lactide-co-glycolide)/chitosan scaffolds," Biomaterials, vol. 30, pp. 6604-6613, 2009.
[46]V. I. Scanga, A. Goraltchouk, N. Nussaiba, M. S. Shoichet, and C. M. Morshead, "Biomaterials for neural-tissue engineering — Chitosan supports the survival, migration, and differentiation of adult-derived neural stem and progenitor cells," Canadian Journal of Chemistry, vol. 88, pp. 277-287, 2010.
[47]S. H. Hsu, G. S. Huang, and F. Feng, "Isolation of the multipotent MSC subpopulation from human gingival fibroblasts by culturing on chitosan membranes," Biomaterials, vol. 33, pp. 2642-2655, 2012.
[48]L. Wang and J. P. Stegemann, "Extraction of high quality RNA from polysaccharide matrices using cetyltrimethylammonium bromide," Biomaterials, vol. 31, pp. 1612-1618, 2010.


連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top