現在的生活中，大多數人忙於工作而沒有注重飲食習慣和運動，因而有許多疾病的產生。腎臟的儲存能力很強，往往要等到損傷極為嚴重，才會有明顯的徵狀。所以初期的徵狀是不可輕忽的，必須要做仔細的檢查以及按照醫生的指示將藥物服用完畢，才不會導致腎臟發炎。在2007年的統計中，腎臟病就已經位居全台第八位，而根據2013年美國USRDS所公布的統計數據顯示，台灣的末期腎臟發生率位已從世界第一名降至世界第三名，但也由於血液透析的方式大大地延長患者的壽命，洗腎的人口不斷的上升。但需要透過血液透析才能過濾血液中的雜質也就代表著腎臟的功能已經不可恢復，並一生都要依靠血液透析機。因此，腎臟的疾病越早發現並定期接受治療，才可能避免面臨洗腎的情況。
本研究希望能夠以微系統晶片實現模擬腎臟免疫反應的體外微環境，使研究更方便且更具有意義。我們利用微機電技術製作一個腎臟免疫分析晶片，並在晶片中加入低矮結構的設計來提高流阻以產生體外微環境所需的濃度梯度擴散。我們先利用三種濃度1 ng/ml、5 ng/ml以及10 ng/ml的大腸桿菌脂多醣(Lipopolysaccharides from Escherichia coli, E.coli LPS)去刺激腎小管上皮細胞(An immortalized proximal tubule epithelial cell line from normal adult human kidney, HK-2)，受到刺激後的HK-2會分泌訊號因子(cytokine)，我們利用ELISA方式來決定最後使用的濃度為10 ng/ml。之後，在完整的實驗中觀察此濃度刺激下所分泌的訊號因子對人類單核球細胞(Human acute monocyte leukemia cell line, THP-1)造成影響，而從此研究中我們看出THP-1細胞在受到刺激後第三小時開始有貼附的情形產生，也就代表人類單核球細胞有形態上的變化並有可能進行防禦的動作，來保護遭受到攻擊的腎臟。而此研究也成功利用結構設計來確保實驗的準確性並提高流阻形成所需的濃度梯度擴散，且此微流體晶片同時具有檢體使用量較少、低成本以及同步多重複實驗的功能。

Nowadays, most people are focusing on their works and ignoring the importance of diet habit and sports, which causes a lot of diseases. The storage capability of kidney is very important to human bodies. However symptoms would appear only when the kidneys get damaged seriously. Therefore, any early symptoms cannot be ignored. It is important to do a careful examination and follow the diagnosis until the body gets totally cured; otherwise it may cause kidney inflammation. In the statistics of 2007, kidney disease was ranked up to number eight in Taiwan. According to the statistical data published by USRDS in 2013, the incidence of end-stage kidney disease has ranked from first place to third place. Although the patients of end-stage kidney disease decreased, the population of hemodialysis still rises up because it can extend the life effectively.
While hemodialysis machines are used to help patients remove the impurities in the blood, it also means that the function of the kidney cannot be repaired, and patients have to rely on it for the rest of their lives. Therefore, an early detection and regular treatments of the kidney disease may avoid the situation of hemodialysis.
The purpose of this master study is to establish a triplicate microfluidic immunoassay system mimicking the kidney immune response in vitro microenvironment for the studies of kidney disease. Therefore, we use MEMS technology to produce a kidney immunoassay chip, combine narrow-gap channel to define the position of the cells, and increase the flow resistance to generate the concentration gradient diffusion. In this research, we use 1 ng/ml, 5 ng/ml and 10 ng/ml three different concentration of Escherichia coli lipopolysaccharide (E.coli LPS) to stimulate renal tubular epithelial cells (HK-2). Then, we use ELISA to detect the cytokine which secret by HK-2 and decide to use 10 ng/ml E.coli LPS in following experiment.
After HK-2 cells are stimulated by 10 ng/ml E.coli LPS, it will secret cytokine to affect the human acute monocyte leukemia cell line (THP-1) and we find out that the THP-1 will be differentiated into macrophage and have some defensive action to protect the HK-2. In this study, our design can ensure the accuracy of the experiments and increase the flow resistance to generate concentration gradient diffusion. This microfluidic chip also has the features of low-dose specimen requirement, low cost and simultaneous bio-experiments operated in an array form.

Abstract 1
摘要 3
目錄 4
圖目錄 7
第一章 緒論 9
1.1 前言 9
1.2 研究動機 11
1.3 研究背景 13
1.3.1 微機電技術與實驗室晶片 13
1.3.2 腎臟 14
1.3.2.1 結構 14
1.3.2.2 功能 15
1.3.2.3 腎元細胞 15
1.3.3腎臟感染及免疫 17
1.4 文獻回顧 19
1.4.1腎臟感染 19
1.4.2 微流體系統產生濃度梯度 19
1.4.3 微流體系統產生濃度梯度在生物研究上之應用 20
1.4.3.1 Christmas tree 濃度梯度產生器 21
1.4.3.2 Y行濃度梯度產生器 22
1.4.3.3 結構或多孔隙材質濃度梯度產生器 23
1.4.3.4 壓力平衡式濃度產生器 24
第二章 系統理論與晶片設計 25
2.1 系統理論 25
2.1.1 擴散理論 25
2.1.2 微尺度下的物質傳遞 27
2.1.2.1 雷諾數 (Reynolds number) 27
2.1.2.2 特徵擴散長度 (characteristic diffusion length) 28
2.2 微流體流阻分析 29
2.2 晶片設計概念 30
2.2.1 載入腎小管上皮細胞以及LPS微流道設計 32
2.2.2 載入人類單核球細胞之微流道設計 33
第三章 微流道晶片製程 34
3.1製作流程 34
3.1.1 微流道製程 34
3.2 製程結果 37
第四章 實驗結果與討論 39
4.1 實驗材料 39
4.1.1腎小管上皮細胞(Human renal proximal tubular cell line, HK-2)培養 39
4.1.2 大腸桿菌脂多醣 (Lipopolysaccharides from E. coli) 40
4.1.3人類單核球細胞(Human acute monocyte leukemia cell line, THP-1)培養 41
4.1.4 膠原蛋白 41
4.2 實驗儀器架設 42
4.2.1 E.coli LPS 使用濃度測試 42
4.2.2 Enzyme-linked immunosorbent assay (ELISA) 43
4.2.3 計數細胞 47
4.2.4腎臟免疫現象測試 48
4.3 實驗結果 49
4.3.1 低矮結構定義細胞位置 49
4.3.2 E.coli LPS 濃度分析 50
4.3.2 濃度擴散現象 54
4.3.3 人類單核球細胞貼附情況 55
第五章 結論 57
文獻參考 58

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