人類胚胎幹細胞在無滋養層及無異種條件的培養下於帶有生長因子的表面進行培養;Human Embryonic Stem Cells Culture on Growth Factor-immobilized Surface under Feeder-free and Xeno-free Conditions

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题名:	人類胚胎幹細胞在無滋養層及無異種條件的培養下於帶有生長因子的表面進行培養;Human Embryonic Stem Cells Culture on Growth Factor-immobilized Surface under Feeder-free and Xeno-free Conditions
作者:	李孟蓓;Li, Meng-Pei
贡献者:	化學工程與材料工程學系
关键词:	人體胚胎幹細胞;生長因子;肝素;多能性之維持;Human embryonic stem cells;Growth factors;Heparin;Maintenance of pluripotency
日期:	2017-08-14
上传时间:	2017-10-27 12:59:16 (UTC+8)
出版者:	國立中央大學
摘要:	本論文的主題為: 「人體胚胎幹細胞在無滋養層及無異種條件的培養下於帶有生長因子的表面進行培養」。研究內容著重於利用已知成分之生醫材料對人體多能性幹細胞做培養。人體多能性幹細胞分為人體胚胎幹細胞以及人體誘導型多能性幹細胞兩大類，由於此等細胞於適當環境下具有優越的繁殖力以及多能性，近年來於再生醫療領域已成為相當有潛力的研究來源。然而，人體多能性幹細胞的培養成本之高，也一直是研究端及應用端面臨的困難；原因歸咎於人體多能性幹細胞生長必要之生長因子(纖維母細胞生長因子及轉化生長因子-β1)，單價相當昂貴且易在溶液中受溫度干擾或變質而降解(需低溫保存)，培養液之頻繁更換致使整體的培養成本居高不下。有鑑於此，本實驗的主軸除了著重於人體多能性幹細胞重要特性之維持，亦致力於培養成本之降低。基於此理念，本實驗發展出一生醫材料，試圖將生長因子吸附於培養皿表面，使生長因子的結構能被固定且穩定於培養皿表面，同時能減低培養液中生長因子的使用量，藉此降低整體培養的成本。實驗中的生醫材料設計運用了知名的蛋白質穩定物質—肝素，肝素藉由化學合成的方式固著於表面，作為生長因子吸附於材料上的媒介。人體胚胎幹細胞(細胞株型號WA09)貼附於帶有肝素及生長因子的培養皿上後，後續使用生長因子減量的培養液對WA09細胞做長期培養。本研究涵蓋了帶有生長因子的培養皿之設計、人體胚胎幹細胞於少量生長因子供應下培養之觀察及長期培養後人體胚胎幹細胞多能特性之鑑定，也於最終結果獲得最適於人體胚胎幹細胞之生醫材料的組成。;In this master thesis, “Human Embryonic Stem Cells Cultivation on Growth Factor-immobilized Surface under Feeder-free and Xeno-free Conditions”, a method was developed for culturing the hPSCs with chemically defined condition. Human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have emerged as prospective resources in regenerative medicine due to their infinite proliferation ability and pluripotency. However, general hPSCs medium containing crucial growth factors, FGF-2 and TGF-β1, which are easily in degradation in solution but are highly contributing to the proliferation and pluripotency of hPSCs, are considerably expensive, which reflects on the high expense of hPSC cultivation. Therefore, a surface immobilized with growth factors was developed not only to stabilize the growth factors on the surface but also to reduce the usage of growth factor in culture medium. In this study, a well-known protein stabilizer, heparin, was chemically immobilized on the surface to serve as binding sites of growth factors for hESC (WA09) cultivation. After absorption of the growth factors on the heparin-immobilized surface, the hESCs were cultured on the modified surface with reduced usage of growth factor in medium for long-term cultivation. It is concluded that the expansion of hESCs (WA09) with decreasing usage of growth factors system containing heparin was successfully developed and a series of pluripotency examinations for hESCs were conducted after long-term cultivation.
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