臺灣博碩士論文加值系統

在本研究中，我們設計和製造了具有流動相的生物反應器，並以此發展三維動態培養系統。藉由冷凍凝膠法，我們製備富含均勻孔洞及高孔隙率的幾丁聚醣支架作為生物反應器中的固定相，接著進行骨母細胞培養。細胞實驗的結果指出，幾丁聚醣支架具有良好的生物相容性。比較二維與三維培養系統，三維系統提供細胞更多的貼附面積。比較靜態與動態培養系統，若流速為2mL/day時，動態培養環境中的細胞呈現最好的細胞活性，然而動態系統中培養液的流量過高時，則會產生剪切應力導致細胞活性降低，流量過低則是無法表現流動特性。
第二部分的研究中，我們以柚皮苷(Naringin)和地塞米松(Dexamethasone)這兩種具有骨誘導性的藥物為標的，測試藥物在靜態與動態培養系統中對細胞影響的差異性。由實驗結果發現，動態系統促進了柚皮苷和地塞米松對細胞的作用，即在相同的藥物濃度下，細胞活性高於靜態系統、細胞骨分化較靜態系統早、細胞成骨時的鈣含量也遠高於靜態系統。在細胞毒性方面，相較於靜態系統，在動態系統中藥物產生毒性的濃度較低。此結果也表示，動態系統所培養的細胞對於藥物的刺激較為敏感。
比起傳統靜態二維培養，本研究開發的三維動態系統能給予骨母細胞更類似體內環境的培養條件，在組織工程方面，更有助於細胞的增生。當應用於藥物測試時，能使藥物對細胞的影響更趨近於人體內的真實情況。

In this research, the three dimensional dynamic culture processes were developed, and the flowing bioreactor was designed and fabricated. Chitosan scaffold in bioreactor was prepared by the freezing-gelation process. The scaffolds showed uniform pores and high porosity. The culture of osteogenic cells on scaffolds was conducted in the static and in the dynamic culture process with controlled flow rates of medium. The result supported that with a suitable range of flow rate, the dynamic system offer the better culture environment than the static cultivation, which revealed by the high cellular viability. However, when the flow rate was higher than 2mL/day, it will generate the shear stress and decrease the cell viability. Oppositely, the flow rate lower than 2ml/day was not effective on
In the second part of this research, two different drugs, Naringin and Dexamethasone, were added into our three-dimensional bioreactor, respectively. From the results, this dynamic system promoted the effect of Naringin and Dexamethasone to cells. In the same condition, the results of cell viability, osteogenic differentiation, and mineralization will be promoted in dynamic system. The results of cytotoxicity also indicate that the cells cultured by dynamic system are more sensitive to drug release.
Compared with two dimensional culture, the three dimensional dynamic system provide the human-like environment for osteogenic cells.

目錄
摘要 V
Abstract VI
圖目錄 X
表目錄 XIII
專有名詞及縮寫 XIV
第一章 緒論 1
第二章 文獻回顧 2
2.1 細胞培養技術 2
2.1.1 細胞三維培養技術與靜態培養系統 2
2.2 細胞動態培養系統 4
2.2.1 批次動態培養系統 4
2.2.2 循環動態培養系統 5
2.2.3 動態系統對骨細胞的影響 7
2.3 藥物於細胞培養中的表現 9
2.3.1 不同系統之藥物表現 9
2.3.2 骨骼相關藥物表現 9
2.3.3 藥物添加於動態系統對於骨細胞影響 13
2.4 骨母細胞分化標記 15
第三章 實驗材料與方法 17
3.1 實驗藥品 17
3.2 實驗儀器 19
3.3 實驗步驟 21
3.3.1 支架的製備 21
3.3.2 動態系統的建立 21
3.4 體外細胞培養 22
3.4.1 實驗操作 22
3.4.2 細胞來源 22
3.4.3 細胞靜態培養 23
3.4.4 細胞播種 23
3.4.5 細胞動態實驗 24
3.4.6 細胞冷凍保存 24
3.4.7 細胞解凍及培養 25
3.4.8 細胞計數 25
3.4.9 粒線體活性測試 26
3.4.10 鹼性磷酸酶測試 28
3.4.11 蛋白質濃度測定 29
3.4.12 電子顯微鏡觀察前細胞樣本處理方式 31
3.4.13 柚皮苷添加 32
3.4.14 地塞米松添加 33
第四章 實驗結果與討論 34
4.1 二維與三維靜態培養的差異 34
4.2 細胞於靜態系統與不同流速之動態系統的差異 37
4.2.1 細胞活性表現 37
4.2.2 細胞型態觀察 40
4.3 柚皮苷於動態與靜態培養系統的差異 44
4.3.1 細胞活性表現 44
4.3.2 細胞型態觀察 46
4.3.3 細胞分化表現 48
4.3.4 細胞成骨表現 51
4.3.5 柚皮苷濃度對於靜態與動態系統之細胞毒性影響 56
4.4 地塞米松於不同培養系統的差異 58
4.4.1 細胞活性表現 58
4.4.2 細胞型態觀察 60
4.4.3 細胞分化表現 62
4.4.4 細胞成骨表現 65
4.4.5 地塞米松濃度對於不同系統之細胞毒性影響 69
第五章 結論 72
參考文獻 73
Appendix 82

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