設計與製作圓錐平板型生物反應器以探討剪應力對大鼠骨髓幹細胞生長與型態之影響

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姓名

馬大翔(Da-Shiang Ma) 查詢紙本館藏

畢業系所

機械工程學系

論文名稱

設計與製作圓錐平板型生物反應器以探討剪應力對大鼠骨髓幹細胞生長與型態之影響
(Study of the Effects of Shear Stresses on the Growth and Morphology of Rat Bone Marrow Stem Cells with the Cone-Plate Bioreactor)

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摘要(中)

本研究設計圓錐平板式生物反應器，利用圓錐旋轉時帶動培養液產生剪應力，對平板上的細胞施予可控制的定量剪應力刺激值。使用此生物反應器，對大鼠骨髓幹細胞進行1天14小時的培養，並對其分別施加1.4、5.5、11、16.4 dyn/cm2的剪應力刺激。結果發現施予剪應力刺激的細胞其生長速度受到抑制，而刺激強度超過1.4 dyn/cm2後，細胞型態會隨著刺激強度增加而逐漸變差，細胞存活數目減少。此外，在1.4 dyn/cm2的4小時刺激強度下，將培養時間延長至3天又14小時，亦發現細胞生長速度受到抑制，且於細胞型態上，出現細胞開始拉長並有方向性排列的情況。
實驗結果顯示，本研究設計的圓錐平板裝置可穩定提供剪應力機械刺激，且能於體外穩定培養細胞。未來，對於經過剪應力刺激已出現形態變化的細胞，可於生化方面進行相關檢測，並給予細胞不同強度或週期性的剪應力刺激，觀察細胞增殖數量的生長變化。

摘要(英)

We designed a cone-plate bioreactor. It produces fluid shear stress by rotating the cone that drives the culture medium to flow in a circular way. Rat bone marrow stem cells were cultured on coverslips placed on the plate. The cells experienced the shear stress of 1.4、5.5、11、16.4 dyn/cm2 respectively for 4 hours in 1 day and 14 hours in the bioreactor.
Results showed the shear stresses changed the morphology and inhibited the cell growth when the shear magnitudes were over 1.4 dyn/cm2. We also found that, by applying shear stress of 1.4 dyn/cm2 for 4 hours within 3 days and 14 hours, the cells became elongated in shape and aligned to each other.
The study demonstrates that the cone-plate bioreactor can provide well-defined stimulus to the cells cultured in vitro. The growth inhibition and shape changes of the cells by shears shall merit further research efforts to classify the underlying mechanisms and biochemical pathways.

關鍵字(中)

★ 幹細胞
★ 圓錐平板裝置
★ 生物反應器
★ 剪應力
★ 組織工程

關鍵字(英)

★ Stem Cells
★ Cone-Plate Bioreactor
★ Bioreactor
★ Shear Stress
★ Tissue engineering

論文目次

中文摘要………………………… І
英文摘要…………………………II
誌謝………………………………Ⅲ
目錄………………………………IV
圖目錄………………………. …VII
第一章緒論
1.1 前言 ………………………………………………………1
1.2 文獻回顧……………………………………………………4
1.2.1 生物反應器與剪應力刺激機械刺激……………………4
1.2.2 剪應力生物反應器………………………………………7
1.3 研究動機……………………………………………………9
第二章生物反應器設計與製作
2.1 設計前言…………………………………………………………………10
2.2 剪應力產生原理以及剪應力計算…………………………10
2.2.1 剪應力產生原理…………………………………………10
2.2.2 定常剪應力計算…………………………………………11
2.3 培養室設計…………………………………………………12
2.3.1 圓錐平板尺寸設計………………………………………12
2.3.2 系統剪應力性能表現……………………………………14
2.4 生物反應器機構設計………………………………………15
2.4.1 培養環境系統……………………………………………17
2.4.2 馬達驅動裝置與馬達箱體………………………………18
2.4.3 承載平板空間……………………………………………25
2.4.4 圓錐平板間距定位調整…………………………………26
第三章實驗方法
3.1 細胞來源……………………………………………………29
3.2 玻片滅菌程序………………………………………………29
3.3 細胞種植……………………………………………………31
3.4 實驗前置準備………………………………………………31
3.5 生物反應器系統培養效能與細胞相容性測試……………33
3.6 實驗步驟……………………………………………………36
3.7 系統操作步驟………………………………………………38
3.8 實驗條件……………………………………………………39
3.9 馬達轉速設定………………………………………………40
3.10 結果分析…………………………………………………40
3.10.1 細胞型態觀察…………………………………………41
3.10.2 細胞數量計數…………………………………………41
第四章結果與討論
4.1 剪應力強度對細胞生長狀態的影響………………………43
4.1.1 剪應力刺激強度：1.4 dyn/cm2............................................44
4.1.2 剪應力刺激強度：5.5 dyn/cm2............................................46
4.1.3 剪應力刺激強度：11 dyn/cm2............................................49
4.1.4 剪應力刺激強度：16.4 dyn/cm2............................................51
4.2 剪應力對細胞型態的… …………………………………54
第五章結論與未來展望………………………………………63
參考文獻..…………………………………………………………………65
附錄
實驗試藥及儀器………………………………………………72

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指導教授

鍾志昂(Chih-Ang Chung)

審核日期

2009-2-2

推文