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研究生： 黃婉棣
論文名稱： 軟骨組織工程： 軟骨化潛力之間葉幹細胞之篩選
Isolation of mesenchymal stem cells with chondrogenesis potential by collagen gel
指導教授： 朱一民
口試委員: 湯學成
姚少凌
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2011
畢業學年度： 99
語文別： 中文
論文頁數： 76
中文關鍵詞： 間葉幹細胞 、篩選 、軟骨化 、組織工程
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    • 間葉幹細胞為多能性細胞，具有分化成許多種類細胞的能力。目前臨床常見之應用為治療白血病之骨髓幹細胞移植。然而間葉幹細胞至今尚未具有一獨特之細胞標幟，只能利用去除其餘細胞之方法取得間葉幹細胞，由此可知間葉幹細胞非一單純種類之細胞。
    　　而本研究著重於利用自製簡單篩選培養皿來篩選間葉幹細胞，其則分為貼附型及懸浮型兩者，並且將兩種細胞群誘導培養後，於不同時間點分析其基因與蛋白表現。由實驗結果可知兩種細胞群經誘導後，貼附型細胞群在軟骨化基因上具有較佳之表現，並分泌較多之GAG，且其細胞型態與軟骨細胞相似。由此推測此篩選系統可將間葉幹細胞篩選出較易形成軟骨結構之細胞群。

    第一章、文獻回顧 1 1.1 組織工程 1 1.2 關節軟骨 3 1.2.1 關節軟骨組織結構 4 1.2.2 細胞外間質 5 1.3 間葉幹細胞 6 1.3.1 成體間葉幹細胞來源 7 1.3.2 間葉幹細胞特性 8 1.3.3 間葉幹細胞在軟骨分化上的相關研究 9 1.3.4 間葉幹細胞之臨床應用 10 1.4篩選系統 11 1.4.1細胞抗原特異性篩選 11 1.4.2 微機電分離系統 12 1.4.3 細胞貼附性篩選 13 1.5 軟骨生醫材料 14 第二章、實驗動機與目的 17 第三章、實驗架構與方法 18 3.1　實驗架構 18 3.2　實驗藥品 19 3.3　實驗儀器 20 第四章、實驗步驟與方法 21 4.1第一型膠原蛋白篩選培養皿配製及使用 21 4.1.1第一型膠原蛋白配製及成膠 21 4.1.2第一型膠原蛋白篩選盤篩選後細胞收集 21 4.2支架製作 22 4.2.1第二型膠原蛋白萃取純化 22 4.2.2第二型膠原蛋白支架之塑成 22 4.2.3第二型膠原蛋白支架交聯及滅菌 23 4.3間葉幹細胞與軟骨細胞培養及支架誘導培養 23 4.3.1骨髓間葉幹細胞培養及繼代 23 4.3.2 支架誘導培養 23 4.4 生物活性分析 24 4.4.1 基因分析 24 4.4.2 GAG測定 25 4.4.3 DNA分析 26 4.5 支架組織切片染色分析 26 4.5.1 HE stain 26 4.5.2 Alcian blue 27 4.5.3 Safranin O 27 4.6 支架特性分析 27 4.6.1 支架特性SEM觀察 27 第五章、結果與討論 29 5.1 誘導用膠原蛋白二型支架 29 5.2 基因分析 30 5.2.1 軟骨化基因分析 30 5.2.2 硬骨化基因分析 38 5.2.3 脂肪化基因分析 42 5.3 GAG/DNA分析測定 44 5.3.1 GAG/DNA分泌量 44 5.3.2 GAG/DNA相對量比較 45 5.4 支架組織切片染色 46 5.4.1 蘇木紫與伊紅染色結果（Hematoxylin and Eosin stains） 46 5.4.2 阿爾襄藍染色結果（Alcian Blue） 55 5.4.3 藏紅（Safranin O）染色結果 64 第六章、結論與未來展望 73 Reference 75

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