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研究生: 林秋雯
論文名稱: 仿肝小葉實驗室晶片檢測酒精中毒造成肝發炎 誘發嗜中性球趨化反應之研究
Lobule-Mimetic Lab Chip for Studying Alcohol Induced Neutrophil Migration in Alcoholic Liver Inflammation
指導教授: 劉承賢
Liu, Cheng-Hsien
口試委員: 盧向成
Lu, Shiang-Cheng
劉承賢
Liu, Cheng-Hsien
彭慧玲
Peng, Hwei-Ling
學位類別: 碩士
Master
系所名稱: 工學院 - 動力機械工程學系
Department of Power Mechanical Engineering
論文出版年: 2012
畢業學年度: 100
語文別: 中文
論文頁數: 58
中文關鍵詞: 肝發炎嗜中性球濃度梯度聲波混合器細胞排列
外文關鍵詞: Liver inflammation, neutrophils, concentration gradient, acoustic mixer, cell pattern
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  • 近年來,肝臟相關的疾病如慢性肝病、肝硬化、肝癌成為臺人死亡十大死因
    之一。相關治療藥物的開發、測試,一直都是醫藥科學目前發展的重點;在研發
    新藥過程因為臨床試驗費用昂貴且相關規定嚴格,因此開發新藥的成功率極低。
    隨著生物微機電技術的發展,可以達成於體外仿肝組織建構,提供有效的進行藥
    物測試,雖然體外測藥與實際應用於體內真實測藥情形仍存在差異性,但可提供
    初步且易於觀察的體外藥物測試反應的平台,此外,相關研究人員亦持續朝著更
    具真實性的體外肝重建模型發展。
    本研究設計一整合型肝藥物檢測平台。利用生醫微機電技術設計仿肝小葉電
    極進行體外肝臟細胞排列,藉提供不同頻率的交流電壓而產生之正負介電泳力,
    控制細胞被極化的程度將細胞排列成仿肝組織之形態並增加細胞和細胞之間的
    交互作用。此外,搭配濃度梯度流道設計與聲波震盪器產生 5 種不同的乙醛濃度
    影響下形成體外促使肝細胞發炎的模型,設計之檢測晶片包含上、下流道,且其
    中間為一層具有直徑 5μm 圓孔的多孔性 PDMS 薄膜,在上流道注入嗜中性白血
    球後,嗜中性球會貼附於 PDMS 薄膜上,接著因下流道體外重建肝發炎生物模
    型之影響而產生趨化反應。實驗結果中可以發現細胞排列成仿肝小葉後,在乙醛
    濃度 175μM 刺激細胞下,比起未進行細胞排列的肝小葉 IL-8 分泌高出 3 倍。嗜
    中性白血球也受肝發炎情形產生不同的趨化數目。
    此研究的目標希望以體外重建工程,建立一仿肝實驗室晶片,使其越接近於
    人體真實肝組織環境,可觀察酒精中毒後,肝發炎生物情形並且期望可以應用於
    藥物檢測的應用中。


    In recent years, liver-related diseases such as chronic liver disease, cirrhosis and liver cancer, are leading cause of disease related deaths Related drug development and testing have always been the goal of medical research. Drug development is expensive due to the several steps involved before the pre-clinical and clinical trials making it expensive and yet the success rate is very low. With the development of biological micro-electromechanical technology, we were able to construct a liver-lobule-mimetic tissue for effective drug testing. Although there are differences in the in vitro testing of drug and it’s in vivo practical application, the in vitro platform provides preliminary and easy observation which can be useful for researchers towards the development of in vitro liver reconstruction model.
    This research presents an integrated drug testing lab chip where in the liver-lobule-mimetic electrode pattern is used to generate in vitro liver cell pattern. Based on polarity difference of cells, positive and negative DEP force is utilized to manipulate and pattern heterogeneous cells for mimicking the inherent hepatic morphology, increasing the cell-cell interaction.
    The concentration gradient generator design and acoustic micromixer generates five different concentrations of acetaldehyde, which is used to induce in vitro liver inflammation and observe neutrophil migration in Alcoholic Liver Inflammation In the design of chip test platform, there are upper channel and bottom channel. Moreover, the intermediate bonding layer of a porous PDMS thin film has a hole which has a diameter of 5μm. When neutrophil injected into upper channel, it would attach to the PDMS thin film. Then bottom channel of chip established in vitro reconstruction of the biological model of liver inflammation to generate chemotactic response. The cell were treated by using acetaldehyde in the concentration of 175μM after the cell were arranged in liver lobule-mimetic. Experimental results indicate that the secretion of IL-8 is 3-fold higher compared with the liver lobule-mimetic without cell arrangement. Furthermore, neutrophil would produce different number of migration by the influence of liver inflammation.
    The ultimate goal of this research is to construct an in vitro liver tissue, mimicking the in vivo liver in its functioning and prove its usefulness in drug testing

    摘要 I Abstract II 目錄 IV 圖目錄 VII 第一章 緒論 1 1.1 實驗室晶片 1 1.2 組織工程 2 1.2.1 肝臟結構與功能介紹 2 1.2.2 肝臟細胞 3 1.3 慢性酒精性肝臟疾病相關研究的重要性 4 1.4 嗜中性球 5 1.5 文獻回顧 6 1.5.1 細胞排列 6 1.5.2 濃度梯度 9 1.5.3 微混合 11 1.5.4 白細胞介素-8 15 第二章 系統架構與分析 16 2.1 PDMS 蝕刻技術 16 2.1.1 乾蝕刻 16 2.1.2 溼蝕刻 16 2.2 微米尺度下的質量傳遞 18 2.3 介電泳 19 2.4 設計概念 21 2.4.1 產生濃度梯度的微流道跟聲波振盪混合器設計 21 2.4.2 仿肝小葉電極設計 21 2.5 數值模值 22 2.5.1 濃度梯度的數值模擬 23 2.5.2 仿肝小葉電極的數值模擬 23 2.5.3 聲波震盪器模擬 24 第三章 微流道製程 26 3.1 製程步驟 26 3.2 微製程結果 30 第四章 實驗結果與討論 32 4.1 聲波震盪器混合實驗 32 4.2 濃度梯度的實驗結果 33 4.3 HL-60細胞培養與分化之類嗜中性球細胞 34 4.4 細胞仿肝小葉排列實驗 35 4.4.1 細胞培養 35 4.4.2 表面改質增加細胞貼附能力 35 4.4.3 儀器架設 35 4.4.4 細胞染色與置換介電泳緩衝液 36 4.4.5 細胞排列過程 37 4.5 薄膜蝕刻結果 38 4.6 IL-8檢測 39 4.7 細胞存活率MTT檢測 40 4.8 晶片外測試肝發炎現象 41 4.9 晶片上測試肝發炎現象 44 4.9.1 無細胞排列肝發炎 44 4.9.2 仿肝小葉細胞排列肝發炎 47 第五章 結論 55 參考文獻 56

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