臺灣博碩士論文加值系統

　　膝關節軟骨細胞在冷凍保存領域研究較少，以往大多是以傳統冷凍保存方式為主，而玻璃化冷凍保存應用在其他細胞較多，但在膝關節軟骨細胞並未發展成熟，玻璃化冷凍保存在其他細胞已經發展一段時間，而玻璃化冷凍保存需藉由高濃度抗凍劑及高冷凍速率才能使細胞液達到玻璃化狀態，因此本研究將會使用實驗室所開發的強制對流玻璃化冷凍保存系統結合低溫液態氧進行玻璃化冷凍保存，並與傳統冷凍保存及原生控制組做對照，觀察存活率及生化特性的表現。
　　為確保強制對流玻璃化冷凍保存系統之冷凍速率可達玻璃化，本研究使用22.5%(v/v)EG + 0.5M sucrose抗凍劑，所測得冷凍速率為29,500 ± 3,000 ℃/min，並由趨勢圖看出無平緩現象即可玻璃化之，並進一步使用豬、人膝軟骨細胞進行玻璃化冷凍保存，其結果豬膝軟骨細胞相對存活率81.65± 3.77%，人膝軟骨相對存活率為68.66%，約高出傳統冷凍4- 10%。在玻璃化冷凍後進行培養觀察生化特性表現，研究結果表示玻璃化冷凍保存之生化特性恢復較快，約莫5-7天可回復一定的生化表現，而傳統冷凍保存恢復力較差。
　　從結果表示本研究所使用的強制對流玻璃化冷凍保存系統可運用在膝關節軟骨細胞上，並有良好存活率及生化表現，提供日後學者研究上之參考。

目錄 IV
第一章 緒論 1
1-1 前言 1
1-2 低溫生物學簡介 5
1-3 化學免疫染色法 10
1-4 文獻回顧 12
1-5 研究動機與目的 22
1-6 研究創新與貢獻 23
1-6-1 研究創新 23
1-6-2 研究貢獻 24
第二章 研究方法 28
2-1 前言 28
2-2 膝軟骨細胞分離 28
2-2-1 實驗材料、設備與化學試藥 28
2-2-2 膝關節軟骨細胞分離步驟 31
2-2-3 膝軟骨細胞存活率、濃度量測 32
2-3 強制對流系統之玻璃化冷凍保存實驗 34
2-3-1 實驗材料、設備與化學試藥 34
2-3-2 階段式抗凍劑添加 36
2-3-3 玻璃化冷凍保存 37
2-3-4 強制對流裝置冷凍速率量測 39
2-4 傳統冷凍保存實驗 39
2-4-1 實驗材料、設備與化學試藥 39
2-4-2 傳統冷凍保存 40
2-5 膝軟骨細胞生化表現 41
2-5-1 實驗材料、設備及化學試藥 41
2-5-2 第二型膠原蛋白染色實驗 43
2-6 免疫細胞化學染色法之螢光分析與校正 45
2-6-1 實驗材料、設備及化學試藥 45
2-6-2 螢光顆粒校正 46
2-6-3 螢光影像拍攝與設定 46
2-6-4 ImageJ 影像處理及分析 47
第三章 實驗結果 59
3-1 前言 59
3-2 強制對流玻璃化冷凍系統之冷凍速率量測及抗凍劑添加程序 59
3-3 膝軟骨細胞冷凍保存之細胞存活率 60
3-4 冷凍保存前後膝關節軟骨細胞之生化特性表現結果 60
第四章 結果與討論 71
4-1 強制對流玻璃化冷凍系統之冷凍速率量測及抗凍劑添加討論 71
4-2 膝軟骨細胞冷凍保存之細胞存活率討論 73
4-3 冷凍保存前後膝關節軟骨細胞之生化特性表現結果討論 74
第五章 結論與未來研究方向 77
5-1 前言 77
5-2 總結 77
5-3 未來研究方向 78
參考文獻 79

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