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研究生:王建智
研究生(外文):Jian-Jhih Wang
論文名稱:微流體晶片中人類初代自然殺手細胞毒殺血癌細胞之即時分析
論文名稱(外文):A Real-Time Investigation on Cytotoxic Assay of Primary Human Natural Killer Cells against Leukemic Cells in Microfluidic Device
指導教授:黃榮山
口試委員:沈弘俊蔡博宇
口試日期:2015-07-13
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:應用力學研究所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:64
中文關鍵詞:初代自然殺手細胞微流體晶片微機電製程即時觀測系統
外文關鍵詞:primary natural killer cellsmicrofluidic deviceMEMSreal-time observation system
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近年來有越來越多以自然殺手細胞療法治療血癌的臨床案例,自然殺手細胞植入治療的副作用小,可以維持患者較好的生活品質。由於每個人的自然殺手細胞毒殺能力不同,選擇高毒殺能力的捐贈者是重要的環節。傳統上常使用流式細胞分析儀檢測細胞的毒殺能力,然而此分析儀需要使用大量細胞(5×105~106),而在臨床實務上,自然殺手細胞數量有限,因此不適合挪移大量細胞做毒殺能力檢測。
本研究利用微機電製程技術製造出一微流體晶片來檢測細胞毒殺能力。透過微流道結構設計以及流體壓力差的控制技術,達到捕捉懸浮性細胞的目的,並搭配細胞螢光標定技術與倒立式螢光顯微鏡,建置出一套即時影像觀測系統。
本實驗利用微流體晶片檢測自然殺手細胞株(NK-92MI)對血癌細胞株(K562)的毒殺能力,改變不同的效能對目標細胞比例(E/T ratio)分別為0.5:1、1:1、2:1,結果顯示當E/T ratio越大時,所得到NK-92MI之毒殺率也會越高。為了證明本研究之微流體晶片可運用於臨床上,本實驗從四位健康捐贈者身上取得初代自然殺手細胞,並隔周進行毒殺率檢測。結果顯示四位捐贈者的自然殺手細胞在體外培養增生14天後,毒殺率已達到飽和狀態,分別是63.29 %,73.90 %、56.38 %與75.68 %。此外,將相同的細胞樣本於流式細胞分析儀進行毒殺率分析,結果顯示晶片與流式細胞分析儀檢測的毒殺率結果相近。
本研究之微流體晶片成功運用少量細胞(101~102)分析出初代自然殺手細胞之毒殺率,且其結果與流式細胞分析儀檢測結果具有一致性。成功克服流式細胞分析儀需要大量細胞(5×105~106)才能檢測的缺點,也證實了本研究之微流體晶片搭配即時觀測系統可取代流式細胞分析儀作為新型毒殺率檢測平台。


In recent years, natural killer cells (NK cells) therapy was used to treat leukemia in increasing clinic cases, due to its less side effects that patients can maintain good quality of life. Since NK cells from different individuals exhibit different performances of cytotoxicity, it is important to choose great cytotoxicity from different donors. In tradition, cytotoxicity detection is performed by using flow cytometry; however, it requires a large numbers of cells (5×105~106) which show a challenge in acquiring primary NK cells of patients in clinic.
In this study, we used MEMS technique to fabricate microfluidic device which allows suspension cells to be trapped by the method of microchannel design and pressure difference manipulation of liquid. By integrating the microfluidic device with fluorescent staining techniques and inverted fluorescent microscope, the real-time observation system was established for the detection of cytotoxicity.
By using the microfluidic device we designed to detect the cytotoxicity of NK cells against leukemia, the result showed that the cytotoxicity is higher when effector to target cell ratio (E/T ratio) is greater. For the present microfluidic device to be used practically in clinic, we obtained primary NK cells from donors, and detect the cytotoxicity once a week. After ex-vivo culture for fourteen days, the cytotoxicity saturated, which perform 63.29 %, 73.90 %, 56.38 %, 75.68 %, respectively. Furthermore, we also compared the cell killing performance of the microfluidic device with flow cytometry, and the results of both methods were comparable.
This study successfully analyzed the cytotoxicity of a few NK cells (101~102) by using microfluidic device. Furthermore, the result of the microfluidic device was consistent with the flow cytometry which commonly require a great number of cells to perform detection.


目錄
誌謝 i
中文摘要 ii
Abstract iii
目錄 v
圖目錄 viii
表目錄 x
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 自然殺手細胞療法 6
1-3.2 自然殺手細胞特性與毒殺機制 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 細胞生物技術介紹 20
2-2.1 細胞培養 20
2-2.2 細胞株與初代細胞培養 22
2-3 細胞骨骼介紹與螢光標定 23
2-3.1 細胞骨骼介紹 23
2-3.2 細胞骨骼螢光染色 25
2-4 細胞凋亡機制與標定 26
2-4.1 細胞凋亡機制 26
2-4.2 細胞凋亡之螢光標定 27
2-5 流式細胞分析儀 28
第3章 研究方法 30
3-1 實驗架構 30
3-2 微流體晶片設計與製程 31
3-2.1 微流體晶片設計 31
3-2.2 微流體晶片母模製程 32
3-2.3 微流體晶片製作整合 35
3-3 細胞培養 38
3-4 細胞樣品製備 40
3-4.1 血癌細胞株K562細胞骨骼螢光標定 40
3-4.2 人類初代自然殺手細胞分離與純化 40
3-4.3 細胞凋亡螢光標定 42
3-5 實驗操作流程 43
3-5.1 微流體晶片毒殺分析 43
3-5.2 流式細胞儀毒殺分析 45
3-6 實驗系統架設 46
第4章 結果與討論 47
4-1 細胞捕捉測試與受力分析 47
4-2 毒殺率計算 50
4-2.1 K562細胞骨骼螢光標定測試 50
4-2.2 微流體晶片與流式細胞分析儀之毒殺率計算 50
4-3 細胞株毒殺測試 52
4-4 人類初代自然殺手細胞毒殺測試 53
第5章 結論與未來展望 59
5-1 結論 59
5-2 未來展望 60
Reference 61


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