臺灣博碩士論文加值系統

白血病又俗稱血癌，主要因人體骨髓造血幹細胞之DNA變異，而過分製造不成熟之白血球，這些不成熟的白血球大量存在於血液中會嚴重影響正常血球的功能。自然殺手細胞植入治療法因為副作用小，可以維持病患相對較良好的生活品質，因此近年來有越來越多的患者選擇使用自然殺手細胞植入療法治療白血病。
共軛焦顯微鏡利用掃描樣本共軛焦面再於電腦重建影像的方式，可得到極高解析度與對比度之螢光影像，為目前最先進之螢光顯微技術。在使用共軛焦顯微鏡分析自然殺手細胞毒殺特性時，傳統上須將細胞固定製成玻片樣本；新型倒立式共軛焦顯微鏡可用以觀察活細胞，然而在觀測懸浮性細胞時，會因細胞的漂動而造成長期細胞追蹤觀測的困難。
本研究使用微機電製程技術製作一微流體生物晶片，藉由微流道設計與微流體操控技術，達到定量捕捉懸浮性細胞的功能。並且整合倒立式高解析度共軛焦顯微鏡與螢光染色技術，架設一套高解析度即時性影像觀測與紀錄系統，可用以分析少量的細胞之反應行為，並做長期的追蹤觀測。在實際應用上，本研究針對自然殺手細胞株(NK-92MI)毒殺血癌細胞株(K562)作分析。首先對細胞晶片之細胞捕捉特性作探討，經過實驗與驗證後選擇適當的流體平均流速(287μm/sec)與細胞捕捉壓力(≤23Pa)等參數。接下來再以螢光染色技術對血癌細胞株的細胞骨骼做標記，並以免疫螢光法標記自然殺手細胞，經過微流體生物晶片定量捕捉效能細胞對目標細胞比(Effector-to-target ratio，E/T ratio)為2:1之自然殺手細胞與血癌細胞後，於共軛焦顯微鏡下作40~90min與90~240min兩段時間的連續觀測紀錄，成功地拍攝到血癌細胞在被自然殺手細胞毒殺之凋亡過程中，細胞骨骼與細胞形態的連續改變，並且利用細胞凋亡試劑Phiphilux對細胞凋亡作染色的確認。
本研究成功結合細胞晶片微流體細胞捕捉技術與倒立式高解析共軛焦顯微鏡，微流體細胞捕捉技術有助於共軛焦顯微鏡於細胞長期反應中提供高解析度影像追蹤紀錄，另外細胞晶片可僅使用少量細胞並於較接近血液流動的環境中作反應，降低毒殺率測試與細胞特性研究的細胞與藥劑使用量，為細胞特性研究創造一個嶄新的平台。

誌謝 I
中文摘要 II
ABSTRACT III
目錄 V
圖目錄 VII
第1章 緒論 1
1.1前言 1
1.2 研究動機 4
1.3 文獻回顧 6
1.3.1 自然殺手細胞特性 6
1.3.2 自然殺手細胞植入療法 7
1.3.3 共軛焦顯微鏡應用於自然殺手細胞毒殺血癌細胞觀測 9
1.3.3 生物晶片細胞操控技術 11
1.4 論文架構 14
第2章 原理 15
2.1 微流道流場理論分析 15
2.1.1 水力聚焦流場分析 15
2.1.2 流道捕捉細胞壓力差與細胞受力分析 17
2.2 細胞株介紹 20
2.2.1 自然殺手細胞株(NK-92MI) 20
2.2.2 血癌細胞株(K562) 20
2.3 細胞骨骼介紹與螢光染色 21
2.3.1 細胞骨骼介紹 21
2.3.2 細胞骨骼螢光染色 22
2.4 細胞凋亡與標定介紹 23
2.5 免疫細胞化學染色技術 25
2.6 共軛焦顯微鏡 26
2.6.1 共軛焦顯微鏡基本原理介紹 26
2.6.2 ZEISS LSM780共軛焦顯微鏡介紹 27
第3章 研究方法 28
3.1 實驗架構 28
3.2 細胞晶片設計與製作 29
3.2.1 細胞晶片設計 29
3.2.2 細胞晶片微流道母模製程 30
3.2.3 細胞晶片製作整合 33
3.3 細胞培養 36
3.3.1細胞株解凍培養 36
3.3.2 NK-92MI自然殺手細胞株培養 36
3.4 細胞樣品製備與實驗操作流程 39
3.4.1 細胞螢光標定 39
3.4.2 實驗操作流程 40
3.5 實驗系統架設 43
第4章 結果與討論 45
4.1 細胞晶片捕捉機制探討 45
4.1.1 主流道流體壓力估算 45
4.1.2 螢光球捕捉測試 47
4.1.3 細胞捕捉測試 48
4.1.4 細胞受力分析 50
4.2 共軛焦顯微鏡觀察分析 51
4.2.1 K562細胞骨骼螢光染色測試 51
4.2.2 NK-92MI細胞螢光標定測試 52
4.2.3 40~90分鐘細胞毒殺反應追蹤觀察 53
4.2.4 90~240分鐘細胞毒殺反應追蹤觀察 54
4.2.5 Phiphilux細胞凋亡螢光標定 55
第5章 結論與未來展望 56
5.1 結論 56
5.2 未來展望 57
參考文獻 58

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