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研究生： 陳彥伶
Chen, Yen-Ling
論文名稱： 微流體晶片結合介電泳細胞排列技術於鉤端螺旋體菌造成腎病變之研究
A Microfluidic Chip Integrated DEP Cell patterning Technology for the Analysis of Leptospirosis on Kidney
指導教授： 劉承賢
Liu, Cheng-Hsien
口試委員: 盧向成
Lu, Hsiang-Cheng
張偉嶠
Chang, Wei-Chiao
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： 2018
畢業學年度： 106
語文別： 中文
論文頁數： 77
中文關鍵詞： 免疫反應 、介電泳 、鉤端螺旋體症 、擴散作用 、膠原蛋白
外文關鍵詞： Immunoassay, Dielectrophoresis, Leptospira, Diffusion, Collagen
相關次數： 點閱：2  下載：0 
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    • 關於腎臟之微流體晶片研究中，主要著重於研究慢性腎臟疾病與離子交換等功能，本研究利用微機電技術製作一微流體晶片，使用固化後具有多孔隙特性的膠原蛋白水膠，以模擬人體腎臟之擴散作用，並結合介電泳技術，以將其應用於研究腎臟細胞與免疫系統之交互作用現象。
    本研究使用鉤端螺旋體(Leptospira)的脂多醣來刺激腎小管上皮細胞(HK-2)，在HK-2受到刺激後，會分泌細胞激素(MCP-1)，此細胞激素以擴散作用傳遞給人類單核球細胞(THP-1)，使其分化，以此驗證其免疫反應路徑。而根據實驗結果顯示，本研究設計之電極使腎臟近曲小管細胞在介電泳操作，並培養48小時後，其存活率依舊大於80%，且不影響其細胞生長型態。而在加入50 ng/ml之鉤端螺旋體脂多醣刺激後，HK-2所分泌之細胞激素(MCP-1)高於其他未經DEP操作之結果。透過此研究之晶片系統實驗，成功於體外建立一仿生之腎臟近曲小管微環境，以研究鉤端螺旋體症造成腎病變之現象。

    Recent studies of kidney-on-a-chip mainly focus on either the chronic kidney disease (CKD) or the function of ion exchange. In this research, MEMS technology was utilized to fabricate the microfluidic chip for kidney in-vitro model and collagen hydrogel with multi-porosity was used for mimicking the diffusion phenomena of human kidney. Furthermore, the microfluidic chip was integrated with dielectrophoresis manipulation to build an immunoassay system and observe the cell-cell interaction.
    In this research, Lipopolysaccharide (LPS) from Leptospira was used to stimulate renal tubular epithelial cells (HK-2). After being stimulated, the HK-2 cells secreted cytokine (MCP-1) transmitted via diffusion to influence the human acute monocyte leukemia cells (THP-1). Then THP-1 cells were observed to differentiate to macrophages. This differentiation verifies the immune response pathway.
    Through the electrode pattern designed in this study, the experimental results showed that the viability of HK-2 cells was still higher than 80% and without any morphological change after DEP operating and 48 hours culturing. After stimulation with 50 ng/ml Leptospira LPS, the cytokine MCP-1 secreted by HK-2 cells was higher than other non-DEP operated results. Through the chip system developed in this research, a microenvironment of proximal tubule has successfully established in vitro to study the leptospiral infection in kidney.

    ABSTRACT----------------------------------------------I 摘要--------------------------------------------------II 致謝--------------------------------------------------III 目錄--------------------------------------------------IV 圖目錄------------------------------------------------VIII 第一章 緒論-------------------------------------------1 1.1 前言-------------------------------------------1 1.2 研究背景---------------------------------------3 1.2.1 生物微機電與實驗室晶片--------------------------3 1.2.2 腎臟------------------------------------------4 1.2.3 鉤端螺旋體症（Leptospirosis)-------------------6 1.2.4 人體免疫系統-----------------------------------8 1.3 研究動機與目的---------------------------------11 1.4 文獻回顧--------------------------------------12 1.4.1 腎臟仿生晶片 (Kidney-on-a-chip)----------------12 1.4.2 濃度梯度於微流體系統----------------------------18 1.4.2.1 Christmas tree濃度梯度產生器--------------------19 1.4.2.2 逐步濃度梯度產生器------------------------------19 1.4.2.3 Y型濃度梯度產生器-------------------------------20 1.4.2.4 結構或多孔隙材質濃度梯度產生器-------------------21 1.4.3 細胞排列技術於微流體系統------------------------22 1.4.4 人類單核性細胞株THP-1細胞-----------------------24 第二章 晶片設計與系統原理-------------------------------26 2.1 微流體系統設計理論-----------------------------26 2.1.1 擴散機制--------------------------------------26 2.1.2 微流體系統之流阻分析---------------------------28 2.1.3 介電泳理論------------------------------------30 2.1.3.1 電泳(Electrophoresis, EP)--------------------30 2.1.3.2 介電泳(Dielectrophoresis, DEP)---------------30 2.2 微流體晶片設計--------------------------------33 2.2.1 系統晶片設計之概念-----------------------------33 2.2.2 電極之設計------------------------------------37 2.2.3 矮結構之設計----------------------------------38 2.2.4 生物激素擴散區之設計---------------------------39 2.3 微流體系統晶片之操作流程-----------------------40 第三章 微流體晶片系統製程------------------------------42 3.1 晶片製作流程----------------------------------42 3.1.1 系統晶片母模之製程流程-------------------------42 3.1.2 電極製程--------------------------------------44 3.1.3 晶片系統之整合製程-----------------------------46 3.2 製程結果--------------------------------------48 第四章 實驗材料與方法----------------------------------49 4.1 材料備置--------------------------------------49 4.1.1 細胞培養--------------------------------------49 4.1.2 膠原蛋白--------------------------------------51 4.1.3 介電泳緩衝液----------------------------------51 4.1.4 細胞螢光染劑----------------------------------52 4.1.5 細胞死活檢測----------------------------------52 4.1.6 酵素結合免疫分析法(ELISA)----------------------53 4.2 實驗與儀器架設---------------------------------54 第五章 實驗結果與討論-----------------------------------55 5.1 刺激實驗參數測試--------------------------------55 5.2 擴散支架測試-----------------------------------56 5.2.1 流場測試--------------------------------------56 5.2.2 膠原蛋白之固化測試-----------------------------57 5.2.3 膠原蛋白之濃度擴散現象-------------------------59 5.2.4 LPS沖洗測試-------------------------------------62 5.3 DEP操作對細胞之探討----------------------------------64 5.3.1 細胞排列----------------------------------------64 5.3.2 死活率測試--------------------------------------66 5.3.3 細胞激素分析----------------------------------67 5.4 免疫細胞型態分析----------------------------------68 第六章 結論與未來展望----------------------------------70 參考文獻----------------------------------------------72

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