臺灣博碩士論文加值系統

由於逐年增加的過敏疾病患者，讓過敏疾病成為重大公共衛生問題。為了節省醫療資源，準確且快速的過敏篩檢為防治的重要課題。本研究探討單一嗜鹼性細胞經被動傳輸與免疫球蛋白IgE接合以及對特定過敏原產生活化作用的機制與電性變化關係。以被動傳輸方式接合免疫球蛋白於嗜鹼性細胞膜表面的蛋白質受體，相較於使用化學物質(例如鈣離子載體)或細胞因子(Cytokine)刺激活化嗜鹼性細胞，則更接近人體過敏機制。
本實驗利用陣列流道配合液動力過濾捕捉單一嗜鹼性細胞。利用液動力過濾捕捉單一細胞的優勢以及特性，包含快速捕捉細胞，提供個別細胞一致性的培養與活化環境。我們製作微陣列電極於捕捉槽，對細胞脫顆粒現象與釋放組織胺機制，進行無標記與非侵入式觀察與阻抗量測分析。實驗發現處於致敏階段的嗜鹼性細胞經塵蟎過敏原(Der p 2)刺激後阻抗大小在高頻(>60 kHz)下降約30%，而相位則是下降約20%。在頻率40 Hz 到100 kHz的量測範圍內可依阻抗值判斷細胞是否活化與產生脫顆粒現象。

Allergic diseases become a major public health threat today as a rapid growth of the patients with such diseases. Fast and accurate detection of allergy plays an important role in prevention and treatment. In this study, basophils were bounded to specific Der p 2 IgE through passive transfer. Then specific antigen was used to stimulate the basophils for observation of the degranulation phenomenon and measurements of impedance response.
The hydrodynamic filtration technique was employed to separate and trap single cells in the microchannels. Advantage and characteristics of the hydrodynamic filtration technique leads to rapid trapping of single cells and consistency of environment for cell culturing. Microelectrodes were fabricated in trapping chambers to measure the impedance of trapped single cells. The impedance measurement offers a label-free and noninvasive means for analysis of cell degranulation and histamine releasing. It is found that a significant downward trend in impedance while sensitized basophils were stimulated by specific antigen. The impedance magnitude and phase of the single basophil decreases approximately 30% and 20, respectively, after the antigen stimulation in the frequency range of 40 to 100 kHz.

致謝 i
摘要 ii
Abstract iii
圖目錄 vi
表目錄 vii
第一章 緒論 1
1.1研究背景與動機 1
1.2 文獻回顧 2
1.2.1經由嗜鹼性細胞所引起的過敏反應 2
1.2.2被動傳輸及其引發的過敏反應 3
1.2.3微流道捕捉單一細胞 7
1.2.4單一細胞阻抗量測 9
1.3研究目標 9
第二章 研究理論與方法 10
2.1 KU812介紹 10
2.2被動傳輸的原理及實驗配置 11
2.3抗體抗原配置 12
2.4細胞培養 14
2.4.1 RPMI培養液配置 14
2.4.2細胞解凍 15
2.4.3細胞繼代 15
2.4.4細胞凍存 16
2.4.5細胞計數 17
第三章 微流體電極晶片設計與製作方法 18
3.1微流道與電極設計 18
3.1.1流道捕捉原理 18
3.1.2分支流量推導 20
3.1.3分支流阻推導 21
3.1.4流道分支長度 22
3.1.5流道結構尺寸 23
3.1.6電極設計 25
3.2微流道與電極裝置製程 26
3.2.1微流道母模微影製程與參數 26
3.2.2軟微影技術 28
3.2.3微電極母模微影製程及參數 29
3.2.4微電極金屬蒸鍍與Lift-off技術 30
3.2.5微流道與微電極對準接合 32
第四章 實驗結果討論 33
4.1細胞阻抗量測示意圖 33
4.2電阻抗量測儀器校正 34
4.3等效電路模型建立與擬合 35
4.4背景溶液量測 38
4.5細胞在不同頻率下響應 41
4.6投入不同濃度過敏原對KU812之影響 41
4.7不同過敏狀態的KU812阻抗變化 47
第五章 結論與建議 50
5.1結論 50
5.2建議 51
六、參考文獻 52
附錄 56
實驗設備與材料 56
不同過敏狀況KU812之等效電路元件擬合參數 61

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