臺灣博碩士論文加值系統

本研究以電化學阻抗量測與螢光影像探討免疫球蛋白E (IgE)介導嗜鹼性細胞株(KU812)，以特異性過敏原之白樺樹花粉刺激細胞之活化反應。本實驗應用電化學阻抗量測，結合三極式電化學生醫晶片，體外檢測嗜鹼性細胞活化反應，定量嗜鹼性細胞活化後釋放的組織胺。同時利用免疫細胞化學技術，以CD63螢光蛋白檢測活化之嗜鹼性細胞膜表面之目標抗原，藉由共軛焦顯微鏡成像判斷嗜鹼性細胞的活化程度。比較電化學阻抗量測之結果與螢光標記檢測活化之量化結果，對與活化孵育環境溫度及投入過敏原濃度之影響呈現一致性的趨勢。電化學阻抗法發現，環境溫度由26℃增加至37℃時阻抗差值(△R)增加幅度可達18%，而螢光標記分析細胞活化比率由29.4%增加至66.9%。過敏原濃度由0.1 μg/ml增至2.5 μg/ml時△R增加幅度可達15%，而螢光標記分析細胞活化比率由18.5%增至80.6%。投入過敏原反應時間(t=5~60 min)與細胞活化比率分析(16%增至65%)具有正相關性，然而從電化學阻抗法分析與投入過敏原反應時間，隨著時間增加因組織胺降解呈下降趨勢(t=5~30 min, △R上升幅度為30%~19%)。

致謝 i
摘要 ii
Abstract iii
目次 iv
圖目次 vii
表目次 ix
第一章 緒論 1
1.1 研究動機 1
1.2 文獻回顧 2
1.2.1 因嗜鹼性細胞引發的過敏反應 2
1.2.2 過敏性炎症介質組織胺 5
1.2.3 過敏免疫檢測與電化學阻抗 7
1.3 研究目標 9
第二章 實驗原理 10
2.1 電化學 10
2.1.1 電子交換與電化學質傳 10
2.1.2 電雙層效應 11
2.1.3 電極量測 12
2.1.4 電化學阻抗 13
2.1.5 阻抗分析電路元件與等效電路 14
2.2 自組裝薄分子膜 18
2.3 抗體與抗原 19
2.4 被動致敏嗜鹼性細胞 19
2.5 免疫螢光 21
2.5.1 抗原標記 21
第三章 實驗方法 23
3.1細胞培養 23
3.1.1 嗜鹼性細胞株KU812 23
3.1.2 細胞培環境 23
3.1.3 細胞凍存與復甦程序 24
3.1.4 細胞繼代 25
3.1.5 細胞計數 25
3.2 細胞致敏化與活化 26
3.2.1 細胞致敏化 26
3.2.2 細胞活化 26
3.3 免疫細胞化學製備與分析 27
3.3.1 共軛焦顯微鏡螢光成像 27
3.3.2 ImageJ 28
3.4 三極式傳感晶片 29
3.5 晶片表面修飾 30
3.5.1 自組裝薄膜程序 30
3.5.2 抗體修飾程序 31
3.6 嗜鹼性細胞釋放之組織胺檢測 32
3.7 電化學阻抗量測 33
3.7.1 等效電路模型 33
3.7.2 傳感晶片量測 34
第四章 結果與討論 35
4.1奈米金電極分析 35
4.1.1 金電極接附BSA確認 35
4.1.2 組織胺之阻抗分析 35
4.2 以電化學阻抗譜分析嗜鹼性細胞活化反應 36
4.2.1 細胞活化釋放介質隨著時間變化之阻抗分析 36
4.2.2 不同過敏原濃度使細胞釋放介質之阻抗分析 37
4.2.3 環境影響細胞活化之阻抗分析 40
4.3 以螢光表面標記物檢測嗜鹼性細胞活化反應 41
4.3.1 活化細胞於不同過敏原濃度之螢光分析 42
4.3.2 過敏原反應時間對細胞活化反應之螢光分析 44
4.3.3 於不同環境活化細胞之螢光分析 46
第五章 結論 48
參考文獻 49
附錄 52
實驗藥品 52
實驗藥品資訊 52
實驗藥品配置 53
實驗設備 54
阻抗分析電路元件 57

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