設計與製作圓錐平板型生物反應器並用以探討流體剪應力對膀胱癌細胞自噬行為之影響

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林楷恒(Kai-Heng Lin) 查詢紙本館藏

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論文名稱

設計與製作圓錐平板型生物反應器並用以探討流體剪應力對膀胱癌細胞自噬行為之影響

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摘要(中)

已有許多研究指出機械力刺激細胞在細胞的型態、功能以及訊息傳遞上扮演重要的角色，而這些機械力同樣也與許多疾病的發展有著重要的關聯。有鑑於物理性刺激研究需要有專門的生物反應器來提供細胞機械力刺激，因此本論文第一部份是以人因工程為導向設計出圓錐平板型生物反應器並用以探討流體剪應力對膀胱癌細胞的影響。此全新圓錐平板型生物反應器特點除了可以提供穩定、均勻的流體剪應力外，也具備維修容易、操作技術門檻低、適用於市售培養皿等優點。從細胞相容性測試、細胞活性分析以及西方墨點法的實驗結果來看，皆證實了本文所設計的生物反應器不但可以作後續的生物實驗分析，也可以重現他人的研究成果，這代表新設計的裝置可以提供穩定的實驗條件。本論文的第二部份為利用此新的生物反應器進行流體剪應力刺激膀胱癌細胞後細胞內之生理反應的研究。由我們的實驗結果發現對膀胱癌細胞株BFTC-905施予12 dynes/cm2 的流體剪應力並刺激24小時，在結合細胞自噬抑制劑的實驗條件下，能拯救細胞的活性；另外，細胞染色螢光訊號的增強也說明了受到流體剪應力刺激過後，細胞會產生自噬行為來回應。透過西方墨點法分析我們觀察到了LC3BII的上調控，Beclin-1、caspase-3、8、9以及PARP於其截切位置均未發現被截切的片段。這些證據證實了承受所施加強度之流體剪應力刺激過後的膀胱細胞會藉由自噬行為嘗試渡過所遇到的逆境，而這段過程並不會誘導細胞凋亡也不會從細胞自噬轉向細胞凋亡，因此本論文認為流體剪應力對於細胞的生理意義為細胞自噬的誘導因子。

摘要(英)

Mechanical force stimulation plays an important role not only in biological morphology, functions and signal transductions but also associated with development of numerous diseases. In order to apply the physical stimulation, the first part of this thesis is based on human factors engineering as a guide to design a cone-plate bioreactor to investigate the shear-stress influence on the stimulated bladder cancer cells. This novel cone-plate bioreactor can provide stable and uniform fluid shear stress and possess advantages such as easy maintenance, low operation barrier and being-ready for commercially available petri dishes. Experiment results of cell compatibility test, cell viability analysis and Western blot verified that this bioreactor not only can be used for subsequent biological experimental analyses. By repeating the research results available in literature we showed this bioreactor can supply stable experimental conditions continuously. The second part of this paper is to study the physiological responses of bladder cancer cells stimulated by fluid shear stresses using this new bioreactor. Experimental results show that when the bladder cancer cell line BFTC-905 was subjected to 12 dynes/cm2 fluid shear stress for 24 hours, the cells’ viability declined. The application of autophagy inhibitor could rescue the cell viability. The enhancement of cell-stained fluorescent signal also illustrated autophagic response occurrence after cells were stimulated by fluid shear stress. Through the Western blot analysis we observed the LC3BII was up-regulated, the cleaved fragments of Beclin-1, caspase-3,8,9 and PARP were not found at its cleaved status. These evidences proved that the bladder cancer cell tried to overcome its adversity by taking the autophagic pathway when subjected to fluid shear stress. The shear stress neither induced apoptosis nor transition from autophagy to apoptosis. We concluded the fluid shear stress was an autophagic inducer for the physiological meaning of cell.

關鍵字(中)

★ 圓錐平板型生物反應器
★ 流體剪應力
★ 膀胱癌細胞
★ 細胞自噬

關鍵字(英)

論文目次

摘要 i
Abstract ii
謝誌 iii
目錄 v
圖目錄 viii
表目錄 xi
第一章緒論 1
1.1 研究動機 1
1.2 文獻回顧 3
1.2.1 膀胱癌與治療方法 3
1.2.2 細胞凋亡與自噬 4
1.2.3 流體剪應力刺激對細胞的影響 5
1.2.4 流體剪應力型生物反應器 7
1.3 研究目的 9
第二章生物反應器設計與製作 13
2.1 設計前言 13
2.2 流體剪應力 16
2.2.1 流體剪應力產生原理 16
2.2.2 流體剪應力計算 16
2.2.3 圓錐設計 18
2.2.4 培養液性質量測 18
2.2.5 系統性能表現 19
2.3 圓錐平板生物反應器機構設計與組裝 19
2.3.1 培養皿治具設計 19
2.3.2 扭力量測機構設計 20
2.3.3 底盤機構設計 22
2.3.4 傳動機構設計 22
2.3.5 圓錐驅動裝置設計 23
2.3.6 扭力量測機構驗證 25
2.4 細胞培養環境系統設計與製作 25
2.4.1 細胞培養條件 26
2.4.2 箱體設計與系統控制迴路 26
2.4.3 均溫性測試 29
第三章實驗方法 49
3.1 細胞培養相關操作 50
3.2 流體剪應力刺激實驗 51
3.3 細胞活性分析 (MTT assay) 52
3.4 螢光染色分析 53
3.5 西方墨點法 (Western Blot analysis) 54
3.5.1 蛋白質定量分析 54
3.5.2 蛋白質電泳 (SDS-PAGE) 55
3.5.3 半濕式 (semi-dry) 蛋白質轉印法 57
3.5.4 免疫染色 57
3.6 統計定量 59
第四章結果與討論 60
4.1 細胞活性分析實驗結果 60
4.1.1 單純流體剪應力刺激 61
4.1.2 流體剪應力刺激結合自噬抑制劑 61
4.1.3 流體剪應力刺激結合凋亡抑制劑 62
4.2 細胞自噬相關之螢光染色分析實驗結果 62
4.3 西方墨點法實驗結果 63
4.3.1 流體剪應力刺激對細胞自噬相關蛋白質的表現量變化 63
4.3.2 流體剪應力刺激對細胞凋亡相關蛋白質的表現量變化 64
4.3.3 流體剪應力刺激對其他蛋白質的表現量變化 64
4.4 討論 65
第五章結論與未來展望 79
參考文獻 82
附錄 90

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指導教授

鍾志昂

審核日期

2017-8-15

推文