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系統識別號 U0026-0812200915062703
論文名稱(中文) 應用共同培養微系統裝置分析剪應力對細胞遷移之研究
論文名稱(英文) Study of cell transmigration using a co-culture microsystem under shear stress
校院名稱 成功大學
系所名稱(中) 工程科學系碩博士班
系所名稱(英) Department of Engineering Science
學年度 97
學期 1
出版年 98
研究生(中文) 蔡身興
研究生(英文) Shen-Hsing Tsai
電子信箱 N9695149@mail.ncku.edu.tw
學號 n9695149
學位類別 碩士
語文別 中文
論文頁數 142頁
口試委員 指導教授-林裕城
口試委員-吳梨華
口試委員-謝清河
口試委員-黃義佑
中文關鍵字 內皮細胞  平滑肌細胞  毛細作用  共同培養  流體剪應力 
英文關鍵字 Smooth muscle cells  Endothelial cells  Fluid shear stress  Co-culture  Capillary 
學科別分類
中文摘要 本研究利用微機電製程製作SU-8微結構,透過複製造模技術製備聚二甲基矽氧烷之微晶片,以應用於內皮細胞與平滑肌細胞之共同培養。研究策略是根據毛細作用原理,將內皮細胞與平滑肌細胞注入該細胞共同培養微晶片中,並分別在特定的培養區內進行精確的分離。另外,設計不同的培養間距與不同大小的外加流體剪應力,以觀察細胞間之交互誘導作用與遷移情況。歸納所有的實驗結果,我們可以得到以下兩點重要結論:(1) 流體剪應力大小對不同的細胞會造成不同的影響,實驗中發現較小的流體剪應力(例如7 dyne/cm2)會抑制平滑肌細胞遷移的速度,但對內皮細胞的影響則較不明顯;而流體剪應力增強時(例如12 dyne/cm2),則以上兩種細胞之遷移速度皆明顯變慢;(2) 培養間距的大小會明顯影響兩細胞間之誘導與遷移,當培養間距較小時(例如50 um與100 um),內皮細胞與平滑肌細胞會互相誘導,進而造成細胞產生遷移現象;而當培養間距較大時(例如200 um與500 um),以上兩種細胞就不會發生互相誘導之情形。本研究所建立的新型平面式細胞共同培養微系統,不但可解決傳統細胞共同培養不容易觀察與混合培養等問題,在未來將有助於生物操控與組織修復等應用領域的發展。
英文摘要 In this study, the SU-8 microstructure and Polydimethylsilcoxane (PDMS) microchip were fabricated by using MEMS and replica molding technologies to co-culture endothelial cells and smooth muscle cells. According to capillary theorem, we drew the endothelial cells and smooth muscle cells in cell co-culture microchip, and separated them into specific culture area accurately. Moreover, the different culture spaces and fluid shear stresses were designed for observing the interaction between cells inducing and the migration. According to the results of this study, we concluded that: (1) the different fluid shear stresses have different effects on various cell. We found that the smaller fluid shear stress (7 dyne/cm2) would inhibit the velocity of smooth muscle cells migration. However, the impact on the endothelial cells is unobvious. The migration velocities of these two kinds of cells are significantly slow for the stronger fluid shear stress (12 dyne/cm2). (2) The size of culture space would interfere with the inducing and the migration of the two kinds of cells. In smaller culture space (50 um and 100 um), endothelial cells and smooth muscle cells would induce mutually, which would further make cell migration; in larger culture space (200 um and 500 um), no inducing reaction took place between endothelial cells and smooth muscle cells. In this study, the novel cell co-culture microsystem could solve the problems of the difficulty of observation and the mix culture. And it will have more advantages in bio-manipulation and tissue repair engineering.
論文目次 摘要 I
ABSTRACT II
誌謝 IV
目錄 V
圖目錄 IX
表目錄 XVIII
第一章 緒論 1
1.1 前言 1
1.2 文獻回顧 3
1.2.1 微圖案於生物培養之技術 4
1.2.2 細胞遷移 11
1.2.3 細胞共同培養技術 15
1.2.4 流體剪應力應用於細胞之研究 18
1.3 研究動機與目的 21
1.4 研究架構 22
第二章 細胞共同培養裝置平台之設計與製作 23
2.1 光罩設計與製作 23
2.2 SU-8製程技術與微結構製作 25
2.2.1 SU-8製程技術 25
2.2.2 微結構製作 26
2.3 PDMS複製模造技術及翻製流程 33
2.3.1 EPOXY二次翻模 36
2.4 細胞共同培養裝置之製作 38
2.5 旋轉式流體裝置平台之設計與製作 39
2.5.1 流體裝置平台之設計 39
2.5.2 流體裝置平台之製作 41
2.5.3 PDMS晶片與轉子載台滅菌 43
第三章 實驗與研究方法 44
3.1 實驗藥品及其配製 44
3.1.1 實驗藥品 44
3.1.2 藥品配製 44
3.2 細胞培養與收集計數 46
3.2.1 細胞繼代培養 48
3.2.2 細胞計數 50
3.3 實驗儀器 54
3.3.1 倒立式螢光光學顯微鏡 54
3.3.2 場放射-掃描式電子顯微鏡 55
3.3.3 表面粗度儀 56
3.4 實驗方法 57
3.4.1 細胞於流體剪應力中之實驗 58
3.4.2 細胞濃度測試之實驗 59
3.4.3 細胞分離共同培養之實驗-無施加流體剪應力下 60
3.4.4 細胞分離共同培養之實驗-施加流體剪應力下 62
第四章 結果與討論 63
4.1 PDMS微結構晶片製程結果 63
4.2 細胞於流體剪應力中之實驗結果 67
4.2.1 流體剪應力之計算 67
4.2.2 流體剪應力對平滑肌細胞之影響 70
4.2.3 流體剪應力對內皮細胞之影響 76
4.3 細胞濃度測試之探討 82
4.4 兩細胞(SMCs & ECs)在不同間距且無施加流體剪應 力下進行共同培養後細胞遷移之結果 85
4.5 兩細胞(SMCs & ECs)在不同間距且施加流體剪應力 下進行共同培養後細胞遷移之結果 94
4.6 統計應用軟體分析 110
4.6.1 細胞在間距500 um下於不同流體剪應力之顯著性 分析 110
4.6.2 細胞在間距200 um下於不同流體剪應力之顯著性 分析 113
4.6.3 細胞在間距100 um下於不同流體剪應力之顯著性 分析 115
4.6.4 細胞在間距50 um下於不同流體剪應力之顯著性 分析 118
第五章 結論與建議 121
5.1 結論 121
5.2 建議 123
參考文獻 124
附錄A 134
自述 142
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