本論文研究設計一仿腎臟近曲小管的實驗室晶片(Lab-on-a-chip)將微流道系統結合介電泳操控平台，應用於鉤端螺旋體菌(Leptospira)造成人體腎病變的研究。在微環境中，將人類單核球細胞(THP-1)受到鉤端螺旋體菌脂多醣刺激後造成的免疫反應訊息傳遞給人類腎臟近曲小管細胞(HK-2)，觀察人類腎臟近曲小管上皮細胞進入上皮細胞間質轉化路徑(EMT)的過程。實驗結果顯示本論文設計之晶片平均能抓取到4×10^6 cells/ml濃度的單核球細胞，受到鉤端螺旋體菌脂多醣(濃度50ng/ml)刺激後，動態培養的晶片環境能提高分泌的細胞激素(IL-1β)，高於靜態培養18%。經由本論文設計的電極圖形使腎臟近曲小管細胞在經由介電泳力排列後，能抓取到2.85×10^7 cells⁄ml濃度的細胞，抓取過後的細胞型態在培養24及48小時後觀察並沒有受介電泳力操作而改變。透過晶片設計以及實驗安排，本論文設計之仿腎臟近曲小管晶片成功建立了體外腎臟近曲小管模型用以研究鉤端螺旋體症造成腎病變的現象。

In this thesis, a proximal tubule-on-a-chip device, which combines a microfluidic system with dielectrophoresis control platform for the study of human nephropathy caused by Leptospirosis, was reported. In this microenvironment on chip, the immune message from human acute monocyte leukemia cells (THP-1) stimulated by Leptospira lipopolysaccharide (LPS) was transmitted to human renal proximal tubular cells (HK-2) and then the epithelial-mesenchymal transition (EMT) phenomena of human renal proximal tubular cells was observed. The experimental results showed that this chip can capture 4×10^6 cells/ml of THP-1, which were stimulated by Leptospira LPS (concentration: 50ng/ml). The dynamic environment increased the secreted cytokine (IL-1β), which was 18% higher than static culture. Through the electrode pattern designed in this paper, the HK-2 could be patterned by the dielectrophoresis force up to the cell concentration of 2.85×10^7 cells⁄ml. Cell morphology was observed after 24 and 48 hours, cells were healthy after dielectrophoretic force operation. The proximal Tubule-on-a-Chip device for leptospiral infection in kidney has been successfully developed and demonstrated through this research. .

Abstract I
摘要 II
致謝 III
目錄 IV
圖目錄 VII
表目錄 X
第1章. 緒論 1
1.1 研究背景 1
1.1.1 生物微機電(BioMEMS)與實驗室晶片(Lab-on-a-Chip) 1
1.1.2 腎臟器官及功能 2
1.1.3 鉤端螺旋體症(Leptospirosis) 3
1.2 研究動機與目的 7
1.3 文獻回顧 9
1.3.1 腎臟仿生晶片(Kidney-on-a-Chip) 9
1.3.2 微流體系統產生濃度梯度之裝置 11
1.3.3 機電系統操控應用於細胞仿生排列技術 17
第2章. 腎臟近曲小管仿生晶片設計與原理 21
2.1 微流體晶片設計理論 21
2.1.1 雷諾數(Reynolds number) 21
2.1.2 微流體系統流阻分析 22
2.1.3 介電泳理論 23
2.1.3.1 電泳 (Electrophoresis) 23
2.1.3.2 介電泳現象（Dielectrophoresis） 24
2.1.3.3 介電泳現象之數學模型 26
2.2 腎臟近曲小管仿生晶片設計 29
2.2.1 腎臟近曲小管仿生晶片設計概念 29
2.2.2 細胞抓取電極裝置設計 33
第3章. 微流體系統晶片製程 36
3.1 製作流程 36
3.1.1 微流道母模製程 36
3.1.2 電極製程 39
3.1.3 腎臟近曲小管仿生晶片製程 40
3.2 製程結果 41
第4章. 實驗材料與方法 43
4.1 材料備置 43
4.1.1 細胞培養 43
4.1.2 介電泳緩衝液(DEP Buffer) 45
4.1.3 晶片操作流程 45
4.2 實驗架設 47
4.2.1 儀器架設 47
4.2.2 酵素結合免疫分析法 (ELISA) 47
4.2.3 螢光免疫分析法(Fluorescence immunoassay) 48
第5章. 實驗結果與討論 50
5.1 孔盤實驗(靜態) 50
5.1.1 單核球細胞(THP-1)細胞濃度以及細菌LPS濃度測試實驗 50
5.1.2 腎臟近曲小管上皮細胞(HK-2)受不同IL-1β刺激濃度測試 56
5.2 仿腎臟近曲小管晶片實驗(動態) 59
5.2.1 中間區域–物質交換區 59
5.2.2 外圍區域-仿微血管區域網絡 64
5.2.3 介電泳力抓取細胞電極裝置實驗 70
第6章. 結論與未來展望 79
參考文獻 80

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