| 研究生: |
劉威宏 Wei-Hung Liu |
|---|---|
| 論文名稱: |
流體剪應力結合1-甲基-3-異丁基黃嘌呤對於人類胎盤幹細胞分化之影響 |
| 指導教授: |
鍾志昂
Chih-ang Chung |
| 口試委員: | |
| 學位類別: |
碩士 Master |
| 系所名稱: |
生醫理工學院 - 生物醫學工程研究所 Graduate Institute of Biomedical Engineering |
| 畢業學年度: | 100 |
| 語文別: | 中文 |
| 論文頁數: | 81 |
| 中文關鍵詞: | 細胞分化 、剪應力 、圓錐平板型生物反應器 、胎盤幹細胞 |
| 外文關鍵詞: | PDMCs, cell differentiation, shear stress, cone-plate bioreactor |
| 相關次數: | 點閱:18 下載:0 |
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目前已有許多研究證實給予幹細胞特定的化學生長因子能誘導幹細胞朝向特定分化之路徑前進,而給予幹細胞不同的物理刺激模式也確實會影響幹細胞基因的表現、蛋白質的分泌及細胞的生長、遷移、增殖、分化等細胞行為。本文主旨為設計製作可對細胞施加流體剪應力刺激之生物反應器。其作動原理為利用馬達轉動並帶動圓錐與平板培養室間之培養液旋轉,而旋轉的培養液會對平板培養室上之細胞進行剪應力刺激,並藉由扭力量測裝置的設計能即時定量監控作用於平板上之剪應力大小與誤差。
經過細胞相容性測試後,本研究以此圓錐平板型生物反應器進行流體剪應力對幹細胞分化影響之研究。嘗試利用1-甲基-3-異丁基黃嘌呤 (1-methyl-3-isobutylxanthine, IBMX) 誘導人類胎盤幹細胞 (Placenta Derived Multipotent Cells, PDMCs) 分化為神經細胞,並於投藥過程中利用自製生物反應器施予1、4、8 dyn/cm2三種不同強度之流場剪應力刺激10分鐘,觀察刺激後2小時與72小時對PDMCs分化之影響。實驗結果顯示IBMX若配合較大之流體剪應力進行PDMCs誘導分化,能增進PDMCs分化為神經細胞之趨勢。
Many research results have demonstrated that stem cells can differentiate into specialized cells by chemical induction. Physical stimuli have also been confirmed to be able to trigger specific gene expressions, induce secretion of proteins and modulate cell behaviors such as growth, migration, proliferation and differentiation.
In this study we designed a cone-plate bioreactor which can produce quantified fluid shear stress. By rotating the cone, the bioreactor drives the culture medium to flow in a circular way, which generates virtually uniform azimuthal shear stress that can be applied to the cultured cells on the plate. In order to perform a real time monitoring over the magnitudes of shear stress, a torque sensor was installed to measure the torque caused by the shear stress on the plate.
After biocompatibility tests, the Placenta Derived Multipotent Cells (PDMCs) were cultured in the bioreactor. The PDMCs were then induced toward neuronal differentiation by applying both the chemical and physical stimulation simultaneously. We applied 1-methyl-3-isobutylxanthine (IBMX) as the chemical agent and investigated the effectiveness of the shear stress on cell differentiation. The application of shear stress along without IBMX addition showed no cell differentiation. However, the PDMCs differentiation initiated by the IBMX were enhanced by simultaneously applying the stress.
Bao, X., Clark, C.B., and Frangos, J.A., 2000. Temporal gradient in shear-induced signaling pathway: involvement of MAP kinase, c-fos, and connexin 43. Am. J Physiol. Heart Circulat. Physiol. 278, p.1598-1605.
Barinaga, M., 2000. Fetal neuron grafts pave the way for stem cell therapies. Science 287, p.1421-1422.
Blackman, B.R., Barbee, K.A., a nd Thibault, L.E., 2000. In vitro cell shearing device to investigate the dynamic response of cells in a controlled hydrodynamic environment. Biomedical Engineering Society 28, p.363-372.
Blackman, B.R, García Cardeña, G., and Gimbrone, M.A., 2002. A new in vitro model to evaluate differential responses of endothelial cells to simulated arterial shear stress waveforms. Journal of Biomechanical Engineering 124, p.397-407.
Breen, L.T., McHugh, P.E., McCormack, B.A., Muir, G., Quinlan, N.J., Heraty, K.B., and Murphy, B.P., 2006. Development of a novel bioreactor to apply shear stress and tensile strain simultaneously to cell monolayers. Review of Scientific Instruments 77, p.104301-104309.
Brignier, A.C. and A.M. Gewirtz, 2010. Embryonic and adult stem cell therapy. Journal of Allergy and Clinical Immunology 125(2), p.336-344.
Brown, T.D., 2000. Techniques for mechanical stimulation of cells in vitro: A review. Journal of Biomechanics 33, p.3-14.
Bussolari, S.R., Dewey, C.F., and Gimbrone, M.A., 1981. Apparatus for subjecting living cells to fluid shear stress. Review of Scientific Instruments 53, p.1851-1854.
Chang S. F., Chang C.A., Lee D.Y., Lee P.L., Yeh Y.M., Yeh C.R., Cheng C.K., Chien S., Chiu J.J, 2008. Tumor Cell Cycle Arrest Induced by Shear Stress: Roles of Integrins and Smad. PNAS 105, p.3927-3932.
Chung, C.A., Tzou, M.R. and Ho, R.W., 2005. Oscillatory flow in a Cone and plate bioreactor. Journal of Biomechanical Engineering 127, p.601-610.
Chung, C.A., Weng, C.S. and Tu, M.Z., 2006. The periodical Shear environment of a cone-and-plate bioreactor. Journal of Fluids Engineering128, p.388-397.
Dawe, G.S., X.W. Tan, and Z.C. Xiao., 2007. Cell migration from baby to mother. Cell Adh Migr1(1), p.19-27.
Dewey, C.F., Bussolari, S.R., Gimbrone, M.A. and Davies, P.F., 1981. The dynamic response of vascular endothelial cells to fluid shear stress. Journal of Biomechanical Engineering 103, p.177-185.
Dong, J.D., Gu, Y.Q., Li, C.M., Wang, C.R., Feng, Z.G., Qiq, R.X., Chen, B., Li, J.X., Zhang, S.W., Wang, Z.G., Zhang, J., 2009. Response of mesenchymal stem cells to shear stress in tissue-engineered vascular grafts. Acta Pharmacologica Sinica, p.530-536.
Ebisawa K, Hata KI, Okada K, Kimata K, Ueda M, Torii S., 2004. Ultrasound enhances transforming growth factor beta-mediated chondrocyte differentiation of human mesenchymal stem cells. Tissue Engineering 10(5-6), p.921–929.
Feugier, P., Black, R.A., Hunt, J.A., How, T.V., 2004. Attachment, morphology and adherence of human endothelial cells to vascular prosthesis materials under the action of shear stress. Biomaterials 26, p.1457-1466.
Furukawa, K., Ushida, T., Sugano, H., Ohshima, N. and Tateishi, T., 1999. Real time observation of platelet adhesion to opaque biomaterial surfaces under shear flow conditions. J. Biomed. Mater. Res 46, p.93–102.
Hsu, S.H., Chen, C.A., 2003. In vitro evaluation of cell loss: retention and repopulation on substrates upon shear flow by a rheometer. Journal of Medical and Biological Engineering 23, p.171-176.
Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM. 2007. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 447, p.879-880.
Langer, R. and Vacanti, J.P., 1993. Tissue Engineering. Science 26, p.920-926.
Lanza, Langer, Vacanti. 2007. Principles of Tissue Engineering 3rd Edition. Elsevier, N.Y., Chapter 1.
Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH. Mar. 2004. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 103, p.1669-1675.
Li G, Zhang XA, Wang H, Wang X, Meng CL, Chan CY, Yew DT, Tsang KS, Li K, Tsai SN, Ngai SM, Han ZC, Lin MC, He ML, Kung HF. 2009. Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow, umbilical cord, and placenta: Implication in the migration. Proteomics 9(1), p.20-30.
Martin, I., Wendt, D., Heberer, M., 2004. The role of bioreactor in tissue engineering. TRENDS in Biotechnology 22, p.80-86.
McBride, S.H, Falls, T., and Knothe Tate, M.L., 2008. Modulation of stem cell shape and fate B: Mechanical modulation of stem cell shape and gene expression. Tissue Engineering 14, p.1573-1580.
Miyanishi K, Trindade MC, Lindsey DP, Beaupré GS, Carter DR, Goodman SB, Schurman DJ, Smith RL., 2006. Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro. Tissue Engineering 12(6), p.1419-1428.
Mooney, M., Ewart, R.H., 1934. The conicylindrical viscometer. Physis 5, p.350-354.
O’Cearbhaill, E.D., Punchard, M.A., Murphy, M. Barry, F.P., McHugh, P.E. Barron, V., 2008. Response of mesenchymal stem cell to the biomechanical environment of the endothelium on a flexible tubular silicone substrate. Biomaterials 29, p.1610-1619.
Ohno M, Gibbons G.H, Dzau V.J, Cooke J.P. 1993. Shear stress elevates endothelial cGMP. Role of a potassium channel and G protein coupling. Circulation.88, p.193–197.
Pelech, I. Shapiro, A. H., 1967, Flexible disk rotating on a gas film next to a wall. Trans. ASME 31, p.577-584
Pörtner, R., Nagel-Heyer, S., Goepfert, C., Adamietz, P., and Meenen, N.M., 2005. Bioreactor design for tissue engineering. Journal of Bioscience and Bioengineering 100, p.235-245.
Rüster, B., Göttig, S., Ludwig, R.J., Bistrian, R., Müller, S., Seifried, E., Gille J. and Henschler, R., 2009. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells. BLOOD 108, p.3938-3944.
Saxena, A.K., 2005. Tissue engineering: Resent concepts and strategies. Journal of Indian Association of Pediatric Surgeons 10, p.14-19.
Scaglione, S., Wendt, D., Miggino, S., Papadimitropoulos, A., Fato, M., Quarto, R., Martin, I., 2007. Effects of fluid flow and calcium phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system. Journal of Biomedical Materials Research A, p.411-419.
Sdougos, H. P., Bussolari, S. R. Dewey, C. F., 1984, Secondary flow and turbulence in a cone-and-plate device. J. Fluid Mech. 138, p.379-404.
Shan Sun YL, Samantha Lipsky, and Michael Cho. 2007. Physical manipulation of calcium oscillations facilitates osteodifferentiation of human mesenchymal stem cells. The FASEB Journal 21, p.1472–1480.
Titushkin I, Sun S, Shin J, Cho M., 2010. Physicochemical Control of Adult Stem Cell Differentiation: Shedding Light on Potential Molecular Mechanisms. Journal of Biomedicine and Biotechnology, p.1-14.
Wang, H., Riha, G.M., Yan, S., Li, M., Chai, H., Yang, H., Yao, Q. and Chen, C., 2005. Shear stress induces endothelial differentiation from a murine embroyonic mesenchymal progenitor cell line. Arterioscler Thrombosis and Vascular Biology 25, p.1817-1823.
Wu C.C., Chao Y.C., Chen C.N., Chien S., Y.C., Chien C.C., Chiu J.J., Yen B.L., 2008. Synergism of biochemical and mechanical stimuli in the differentiation of human placenta derived multipotent cells into endothelial cells. Journal of Biomechanics 41(4), p.813-821.
Yen BL, Huang HI, Chien CC, Jui HY, Ko BS, Yao M, Shun CT, Yen ML, Lee MC, Chen YC., 2005. Isolation of multipotent cells from human term placenta. Stem Cells 23(1), p.3-9.
Yen BL, Chien CC, Chen YC, JT Chen, Huang JS, Lee FK, Huang HI., 2008. Placenta derived multipotent cells differentiate into neuronal and glial cells in vitro. Tissue Engineering Part A 14(1), p.9-17.
蔡瑞芳,2004,「幹細胞與組織工程」,後基因體時代之生物技術,第十九章;281 - 294。
呂明憲,2005,週期式圓錐平板裝置之設計與量測,國立中央大學機械工程學系碩士論文。
王文甫,2008,圓錐平板型生物反應器之設計與製作,國立中央大學機械工程學系碩士論文。
幹細胞與組織工程 教學資源中心,2008,「幹細胞學」。
幹細胞與組織工程 教學資源中心,2008,「再生醫學」。
馬大翔,2009,設計與製作圓錐平板型生物反應器以探討剪應力對大鼠骨隨幹細胞生長與型態之影響,國立中央大學機械工程學系碩士論文。
劉鉉志,2010,設計與製作圓錐平板型生物反應器探討剪應力對幹細胞生長與型態之影響,國立中央大學機械工程學系碩士論文。