(18.204.2.190) 您好!臺灣時間:2021/04/22 07:49
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:宋秋慧
研究生(外文):Chiu-hui Sung
論文名稱:缺氧狀態於材料下對誘導性多功能幹細胞於分化心肌細胞之研究
論文名稱(外文):Differentiation of induced pluripotent stem cells in different materials into myocardial cells under hypoxia condition
指導教授:鍾次文
指導教授(外文):Taz-wen Chung
學位類別:碩士
校院名稱:國立雲林科技大學
系所名稱:化學工程與材料工程系碩士班
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:45
中文關鍵詞:肝素透明質酸明膠缺氧環境誘導性多功能幹細胞
外文關鍵詞:GelatinHeparinHAiPShypoxia condition
相關次數:
  • 被引用被引用:0
  • 點閱點閱:223
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:3
  • 收藏至我的研究室書目清單書目收藏:0
本研究擬使用缺氧狀態來刺激誘導性多功能幹細胞,藉由材料生物可降解性聚己內酯與具有良好生物相容性材料如明膠、肝素、幾丁聚醣與透明質酸等,再與生長因子BMP4與材料Ascorbic acid,以促進誘導性多功能幹細胞分化成心肌細胞之效果。
由實驗結果顯示,已完成PCL材料表面的化學改質,將Gelatin 、Heparin、 HA及CS以不同材料組合於PCL材料上。由靜態接觸角判斷親水性材料如:HA 及Heparin接觸角較低,也可用ATR-FTIR之官能基判斷,可得知各材料已成功交聯於PCL材料上。而體外細胞實驗的部份,材料對於細胞貼附及增生部分還未可得知,但分化的部分,根據初步實驗結果,由免疫螢光染色結果可知,材料部份Heparin與HA的PCL材料確實可使心肌蛋白表現量高於明膠;而iPS細胞在正常培養與缺氧環境下培養,由結果可得知,缺氧狀態亦可使iPS分化成心肌細胞。
This study is to promote induced pluripotent stem cells to differentiate into cardiomyocytes by using hypoxia condition with biodegradable materials polycaprolactone (PCL) and having the biocompatible material such as gelatin, heparin, chitosan and hyaluronic acid and BMP4 growth factors and materials Ascorbic acid.
The experimental results show that the surface has been completely PCL material chemically modified, with Gelatin, Heparin, HA and CS in different material combinations on the PCL material. According to the contact angle of hydrophilic materials such as: the contact angle of HA and Heparin is lower than PCL, and also the ATR-FTIR functional group is used to know the materials having been successfully cross-linked on the PCL material. In vitro cell experiments, the relationships between the materials and cell adhesion and proliferation have not been defined yet. In differentiation part, according to the preliminary results by immunofluorescent staining, the group of Heparin with HA and PCL material does enable myocardial protein expression which was higher than gelatin; it is found that hypoxia condition can promote induced pluripotent stem cells into cardiomyocytes compared to the group cultured under normal condition.
中文摘要 i
Abstract ii
目錄 iii
圖目錄 vi
表目錄 vii
第一章 緒論 1
1-1 生醫材料 2
1-1-1 聚己內酯(poly(ɛ-caprolactone)) 2
1-1-2 明膠(Gelatin) 3
1-1-3 肝素(Heparin) 3
1-1-4 幾丁聚醣(Chitosan, CS) 5
1-1-5 透明質酸(Hyaluronic Acid, HA) 6
1-2 缺氧(Hypoxia) 7
1-3 誘導性多功能幹細胞(Induced Pluripotent Stem Cell ) 8
1-4 研究動機與目的 9
第二章 研究內容與方法 10
2-1 實驗藥品 10
2-2 實驗設備 12
2-3 實驗流程 13
2-4 材料製備 14
2-4-1 PCL film製備 14
2-4-2 Gelatin/PCL、Heparin -Gelatin /PCL、CS-Gelatin /PCL
與HA-Gelatin /PCL之製備 14
2-4-3 HA-Heparin- Gelatin /PCL與HA-CS- Gelatin /PCL之
製備 15
2-4-4 實驗材料之縮寫 15
2-5 材料性質分析 15
2-5-1 接觸角量測 15
2-5-2 傅立葉轉換紅外線光譜儀(ATR-FTIR) 15
2-5-3 TBO Test 16
2-6 細胞培養實驗 16
2-6-1 ips利用懸浮培養成胚胎球 16
2-6-2 體外細胞培養 17
2-6-3 細胞活性測試-MTS 17
2-6-4 細胞於材料貼附及增生之情況-Live&; Dead 染色 17
2-7 體外細胞分化實驗 17
2-7-1 免疫螢光染色 17
2-7-2 即時聚合酶鏈鎖反應Real time-PCR 18
第三章 結果與討論 19
3-1 材料性質分析
3-1-1 接觸角量測 19
3-1-2 傅立葉轉換紅外線光譜儀(ATR-FTIR) 20
3-1-3 TBO Assay 22
3-2 體外細胞培養實驗
3-2-1 細胞培養 23
3-2-2 ips利用懸浮培養成胚胎球 24
3-2-3 細胞活性測試-MTS 25
3-2-4 分化細胞之免疫螢光染色 26
3-2-5 螢光強度之分析 30
3-2-6 RT-PCR 31

第四章 結論 33
參考文獻 34
[1] R.P. Lanza, R. Langer and J. Vancanti, editors. Principles of tissue engine- eering.2nd edition, Academic Press, 2000.
[2] Cai Q, Bei J, Wang S. Synthesis and degradation of a tri-component copolymer derived from glycolide, L-lactide, and epsilon-caprolactone. Journal of biomaterials science Polymer edition. 2000;11:273-88.
[3] Coombes AG, Verderio E, Shaw B, Li X, Griffin M, Downes S. Biocomposites of non-crosslinked natural and synthetic polymers. Biomaterials. 2002;23:2113-8.
[4] Ural E, Kesenci K, Fambri L, Migliaresi C, Piskin E. Poly(D,L-lactide/epsilon-caprolactone)/hydroxyapatite composites. Biomaterials. 2000;21:2147-54.
[5] Hao J, Liu Y, Zhou S, Li Z, Deng X. Investigation of nanocomposites based on semi-interpenetrating network of [l-poly (ε-caprolactone)]/[net-poly (ε-caprolactone)] and hydroxyapatite nanocrystals. Biomaterials. 2003;24:1531-9.
[6] Zhu Y, Gao C, Shen J. Surface modification of polycaprolactone with poly(methacrylic acid) and gelatin covalent immobilization for promoting its cytocompatibility. Biomaterials. 2002;23:4889-95.
[7] Cheng Z, Teoh S-H. Surface modification of ultra thin poly (ε-caprolactone) films using acrylic acid and collagen. Biomaterials. 2004;25:1991-2001.
[8] Dai NT, Williamson MR, Khammo N, Adams EF, Coombes AG. Composite cell support membranes based on collagen and polycaprolactone for tissue engineering of skin. Biomaterials. 2004;25:4263-71.
[9]Tomihata K, Burczak K, Shiraki K, Ikada Y. Cross-Linking and Biodegradation of Native and Denatured Collagen. Polymers of Biological and Biomedical Significance: American Chemical Society; 1993. p. 275-86.
[10] Vohra R, Drury JK, Shapiro D, Shenkin A, Pollock JG. Sealed versus unsealed knitted Dacron prostheses: a comparison of the acute phase protein response. Annals of vascular surgery. 1987;1:548-51.
[11] Jensen N, Lindblad B, Dougan P, Bergqvist D. Collagen and gelatin-impregnated vascular grafts--is their thrombogenicity enhanced? An experimental study. VASA Zeitschrift fur Gefasskrankheiten. 1992;21:365-8.
[12] Lacroix H, Boel K, Nevelsteen A, Suy R. Early inflammatory response to gelatin- and collagen-sealed Dacron prostheses. Annals of vascular surgery. 1995;9:152-4.
[13] Olsen D, Yang C, Bodo M, Chang R, Leigh S, Baez J, Carmichael D, Perala M, Hamalainen ER, Jarvinen M, Polarek J. Recombinant collagen and gelatin for drug delivery. Adv Drug Deliv Rev. 2003;55:1547-67.
[14] Marui A, Tabata Y, Kojima S, Yamamoto M, Tambara K, Nishina T, Saji Y, Inui K, Hashida T, Yokoyama S, Onodera R, Ikeda T, Fukushima M, Komeda M. A novel approach to therapeutic angiogenesis for patients with critical limb ischemia by sustained release of basic fibroblast growth factor using biodegradable gelatin hydrogel: an initial report of the phase I-IIa study. Circulation journal : official journal of the Japanese Circulation Society. 2007;71:1181-6.
[15] Huang S, Zhang Y, Tang L, Deng Z, Lu W, Feng F, Xu X, Jin Y. Functional bilayered skin substitute constructed by tissue-engineered extracellular matrix and microsphere-incorporated gelatin hydrogel for wound repair. Tissue engineering Part A. 2009;15:2617-24.
[16] E. D. Korn, Method of biochemical analysis, Interscience publisher, N. Y. London, 1959
[17] Sundheim G, Bengtsson-Olivecrona G. Lipolysis in Milk Induced by Cooling or by Heparin: Comparisons of Amount of Lipoprotein Lipase in the Cream Fraction and Degree of Lipolysis. Journal of Dairy Science. 1985;68:589-93.
[18] 林孟賢、曾春典(1995):肝素及其在抗凝血治療的應用。當代醫學雜誌,22(1),2-9。
[19] Percival, E. G. V. (1965). Structural Carbohydrate Chemistry. Taipei, Zhuang yuan, pp.302-326, 1965
[20] Denizli A. Heparin-immobilized poly(2-hydroxyethylmethacrylate)-based microspheres. Journal of Applied Polymer Science. 1999;74:655-62.
[21] Beena MS, Chandy T, Sharma CP. Heparin immobilized chitosan--poly ethylene glycol interpenetrating network: antithrombogenicity. Artificial cells, blood substitutes, and immobilization biotechnology. 1995;23:175-92.
[22] Lee J, Yoo JJ, Atala A, Lee SJ. Controlled heparin conjugation on electrospun poly(epsilon-caprolactone)/gelatin fibers for morphology-dependent protein delivery and enhanced cellular affinity. Acta Biomater. 2012;8:2549-58.
[23] Chuang WY, Young TH, Yao CH, Chiu WY. Properties of the poly(vinyl alcohol)/chitosan blend and its effect on the culture of fibroblast in vitro. Biomaterials. 1999;20:1479-87.
[24] Chung TW, Liu DZ, Wang SY, Wang SS. Enhancement of the growth of human endothelial cells by surface roughness at nanometer scale. Biomaterials. 2003;24:4655-61.
[25] Yang MC, Wang SS, Chou NK, Chi NH, Huang YY, Chang YL, Shieh MJ, Chung TW. The cardiomyogenic differentiation of rat mesenchymal stem cells on silk fibroin-polysaccharide cardiac patches in vitro. Biomaterials. 2009;30:3757-65.
[26] Dai NT, Williamson MR, Khammo N, Adams EF, Coombes AG. Composite cell support membranes based on collagen and polycaprolactone for tissue engineering of skin. Biomaterials. 2004;25:4263-71.
[27] Chang CH, Liu HC, Lin CC, Chou CH, Lin FH. Gelatin-chondroitin-hyaluronan tri-copolymer scaffold for cartilage tissue engineering. Biomaterials. 2003;24:4853-8.
[28] Maniwa S, Ochi M, Motomura T, Nishikori T, Chen J, Naora H. Effects of hyaluronic acid and basic fibroblast growth factor on motility of chondrocytes and synovial cells in culture. Acta orthopaedica Scandinavica. 2001;72:299-303.
[29] Haider AS, Grabarek J, Eng B, Pedraza P, Ferreri NR, Balazs EA, Darzynkiewicz Z. In vitro model of "wound healing" analyzed by laser scanning cytometry: accelerated healing of epithelial cell monolayers in the presence of hyaluronate. Cytometry Part A : the journal of the International Society for Analytical Cytology. 2003;53:1-8.
[30] Peattie RA, Nayate AP, Firpo MA, Shelby J, Fisher RJ, Prestwich GD. Stimulation of in vivo angiogenesis by cytokine-loaded hyaluronic acid hydrogel implants. Biomaterials. 2004;25:2789-98.
[31] Drubaix I, Legeais JM, Mounier F, Briat B, Robert L, Renard G. Quantification and localization of hyaluronan in a PTFE polymer implanted in the corneal stroma. Journal of biomedical materials research. 1998;40:442-8.
[32] Morrison SJ, Csete M, Groves AK, Melega W, Wold B, Anderson DJ. Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2000;20:7370-6.
[33] Studer L, Csete M, Lee SH, Kabbani N, Walikonis J, Wold B, McKay R. Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2000;20:7377-83.
[34] Adelman DM, Maltepe E, Simon MC. Multilineage embryonic hematopoiesis requires hypoxic ARNT activity. Genes &; development. 1999;13:2478-83.
[35] Ramirez-Bergeron DL, Runge A, Dahl KD, Fehling HJ, Keller G, Simon MC. Hypoxia affects mesoderm and enhances hemangioblast specification during early development. Development (Cambridge, England). 2004;131:4623-34.
[36] Ezashi T, Das P, Roberts RM. Low O2 tensions and the prevention of differentiation of hES cells. Proceedings of the National Academy of Sciences of the United States of America. 2005;102:4783-8.
[37] Semenza GL. Hypoxia-inducible factor 1: master regulator of O2 homeostasis. Current opinion in genetics &; development. 1998;8:588-94.
[38] Jiang BH, Zheng JZ, Leung SW, Roe R, Semenza GL. Transactivation and inhibitory domains of hypoxia-inducible factor 1alpha. Modulation of transcriptional activity by oxygen tension. The Journal of biological chemistry. 1997;272:19253-60.
[39] Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD, Semenza GL. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Molecular and cellular biology. 1996;16:4604-13.
[40] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663-76.
[41] Lee CT, Huang CP, Lee YD. Biomimetic porous scaffolds made from poly(L-lactide)-g-chondroitin sulfate blend with poly(L-lactide) for cartilage tissue engineering. Biomacromolecules. 2006;7:2200-9.
[42] Amabile G, Meissner A. Induced pluripotent stem cells: current progress and potential for regenerative medicine. Trends in Molecular Medicine. 2009;15:59-68.
[43] Liu H, Yin Y, Yao K, Ma D, Cui L, Cao Y. Influence of the concentrations of hyaluronic acid on the properties and biocompatibility of Cs-Gel-HA membranes. Biomaterials. 2004;25:3523-30.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔