流體剪應力結合1-甲基-3-異丁基黃嘌呤對於人類胎盤幹細胞分化之影響

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姓名

劉威宏(Wei-Hung Liu) 查詢紙本館藏

畢業系所

生物醫學工程研究所

論文名稱

流體剪應力結合1-甲基-3-異丁基黃嘌呤對於人類胎盤幹細胞分化之影響

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摘要(中)

目前已有許多研究證實給予幹細胞特定的化學生長因子能誘導幹細胞朝向特定分化之路徑前進，而給予幹細胞不同的物理刺激模式也確實會影響幹細胞基因的表現、蛋白質的分泌及細胞的生長、遷移、增殖、分化等細胞行為。本文主旨為設計製作可對細胞施加流體剪應力刺激之生物反應器。其作動原理為利用馬達轉動並帶動圓錐與平板培養室間之培養液旋轉，而旋轉的培養液會對平板培養室上之細胞進行剪應力刺激，並藉由扭力量測裝置的設計能即時定量監控作用於平板上之剪應力大小與誤差。
經過細胞相容性測試後，本研究以此圓錐平板型生物反應器進行流體剪應力對幹細胞分化影響之研究。嘗試利用1-甲基-3-異丁基黃嘌呤 (1-methyl-3-isobutylxanthine, IBMX) 誘導人類胎盤幹細胞 (Placenta Derived Multipotent Cells, PDMCs) 分化為神經細胞，並於投藥過程中利用自製生物反應器施予1、4、8 dyn/cm2三種不同強度之流場剪應力刺激10分鐘，觀察刺激後2小時與72小時對PDMCs分化之影響。實驗結果顯示IBMX若配合較大之流體剪應力進行PDMCs誘導分化，能增進PDMCs分化為神經細胞之趨勢。

摘要(英)

Many research results have demonstrated that stem cells can differentiate into specialized cells by chemical induction. Physical stimuli have also been confirmed to be able to trigger specific gene expressions, induce secretion of proteins and modulate cell behaviors such as growth, migration, proliferation and differentiation.
In this study we designed a cone-plate bioreactor which can produce quantified fluid shear stress. By rotating the cone, the bioreactor drives the culture medium to flow in a circular way, which generates virtually uniform azimuthal shear stress that can be applied to the cultured cells on the plate. In order to perform a real time monitoring over the magnitudes of shear stress, a torque sensor was installed to measure the torque caused by the shear stress on the plate.
After biocompatibility tests, the Placenta Derived Multipotent Cells (PDMCs) were cultured in the bioreactor. The PDMCs were then induced toward neuronal differentiation by applying both the chemical and physical stimulation simultaneously. We applied 1-methyl-3-isobutylxanthine (IBMX) as the chemical agent and investigated the effectiveness of the shear stress on cell differentiation. The application of shear stress along without IBMX addition showed no cell differentiation. However, the PDMCs differentiation initiated by the IBMX were enhanced by simultaneously applying the stress.

關鍵字(中)

★ 細胞分化
★ 剪應力
★ 圓錐平板型生物反應器
★ 胎盤幹細胞

關鍵字(英)

★ PDMCs
★ cell differentiation
★ shear stress
★ cone-plate bioreactor

論文目次

中文摘要 I
Abstract II
致謝 III
目錄 IV
圖目錄 VII
表目錄 IX
第一章緒論 1
1.1前言 1
1.2文獻回顧 4
1.2.1人類胎盤幹細胞 4
1.2.2剪應力生物反應器 5
1.2.3化學刺激與物理刺激對於幹細胞之影響 6
1.3研究動機 8
1.4論文架購 9
第二章生物反應器設計與製作 10
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 11
2.2.2定常轉速剪應力計算 12
2.3 培養室設計 13
2.3.1培養液性質量測 13
2.3.2圓錐與平板培養室之設計 14
2.3.3系統剪應力表現 16
2.4生物反應器機構設計 17
2.4.1培養環境系統 18
2.4.2馬達驅動裝置與馬達密封組件 19
2.4.3馬達承載箱體空間 24
2.4.4扭力量測裝置校正 28
2.4.5圓錐與平板培養室間距定位調整 30
第三章實驗方法 33
3.1 細胞來源 33
3.1.1細胞培養 33
3.2玻片滅菌消毒程序 34
3.3實驗前置準備 35
3.4細胞種植 36
3.5生物反應器細胞相容性測試 38
3.6實驗步驟 39
3.6.1人類胎盤幹細胞之種植 39
3.6.2種植細胞計數與型態觀察 41
3.6.3實驗組移入平板培養室 41
3.6.4生物反應器操作步驟 41
3.7實驗條件 42
3.7.1馬達轉速設定 43
3.7.2神經誘導化學藥物濃度配置 44
3.8結果分析 45
3.8.1細胞型態觀察 45
3.8.2免疫螢光染色法 45
3.8.3細胞影像處理與分析 47
第四章實驗結果與討論 48
4.1實驗前言 48
4.2細胞相容性 49
4.3結合IBMX與流體剪應力之分化結果 52
4.3.1負向控制 52
4.3.2正向控制 54
4.3.3單純流場剪應力刺激 56
4.3.4流場剪應力刺激結合IBMX 60
4.4細胞數量與分化率 64
4.4.1細胞數量 64
4.4.2細胞分化率 66
4.5分化結果討論 69
第五章結論與未來展望 71
參考文獻 73
附錄-實驗藥品與儀器 78

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指導教授

鍾志昂(Chih-ang Chung)

審核日期

2012-4-19

推文