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研究生： 卓宛瑩
Cho, Wanying
論文名稱： 快速免疫螢光染色暨細胞自組裝排列之二合一晶片應 用於檢測循環腫瘤癌細胞
The Dynamic Staining Self-Assembly Cell Array (DS-SACA) Chip of Rapid Detection of Circulating Tumor Cells
指導教授： 曾繁根
Tseng, Fan-Gang
口試委員: 王本誠
Wang, Pen-Cheng
楊智勇
Yang, Chih-Yung
學位類別： 碩士
Master
系所名稱： 原子科學院 - 工程與系統科學系
Department of Engineering and System Science
論文出版年： 2019
畢業學年度： 107
語文別： 中文
論文頁數： 77
中文關鍵詞： 循環腫瘤癌細胞 、動態染色 、診斷 、免疫螢光
外文關鍵詞： CTC, dynamic stating, diagonose, immunofluorescence
相關次數： 點閱：3  下載：0 
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    • 台灣十大死因之首已由癌症蟬聯第36年，隨著癌症的死亡時鐘不停地撥快，
    現階段癌症在治療上的高困難度包含：癌細胞的轉移以及使用抗癌藥物後續產生
    的抗藥性及變異型，因此能快速檢測出癌細胞並使其用於後續藥物測試便是本研
    究提出之晶片的設計目標。
    當癌細胞在轉移的過程中，血液中可以檢測出循環腫瘤細胞（Circulating
    Tumor Cell, CTC），循環腫瘤癌細胞是指從原生腫瘤或轉移性腫瘤脫離並進入血
    液循環系統的腫瘤細胞。在檢測上，非侵入式的液態檢測便利且接受度廣。先前
    的研究已顯示CTC的多寡與病人的存活率和病情的預測有正相關，除此之外，現
    階段趨勢不僅要能快速檢測出CTC數量，保留其活性，用於後續培養、並做藥物
    測試才是首要關鍵。目前應用於CTC檢測上最流行的技術為唯一通過FDA 認證的
    CELLSEARCH®系統。然而，此系統檢測時間冗長之餘，會造成細胞的死亡及凋零，
    不僅在檢測過程中極易因細胞的堆疊而導致計數上的誤判，在細胞捕獲培養部分
    也無法做出貢獻，目前這項系統公司已停售。
    本研究旨在提出具有兩種重要功能之DS-SACA(Dynamic Staining
    Self-Assembly Cell Array) 晶片取代離心機，使用新型溫和的染色方式完成免
    疫螢光染色標定的程序，改善傳統方式中細胞因離心而導致操作上地流失；同時，
    在螢光顯微鏡的觀測下，能使細胞自組裝產生單層緊密的陣列。單一晶片便能保
    持細胞的活性、使影像易於判讀細胞形貌並可捕捉珍貴的CTC影像，後端技術搭
    配本實驗室研發的細胞針抓取技術進行捕獲CTC，進行後續的培養及檢測，在個
    人化醫療領域做出貢獻。

    The progression of cancer metastasis creates great difficulty in its treatment.
    Dissemination of cancer occurs by circulating tumor cells (CTC) shed in the vasculature. CTC is one of potential liquid biopsies which may be extremely useful in cancer diagnosis. Studies show the amount of CTC is correlated to overall survival
    and disease-free survives.
    The CellSearch system which is FDA approved assay for CTC detection is applied in clinical use. However, it is lengthy along with inclusion of centrifugal force
    harmful for cell viability. In addition, cells stack up randomly which lead to the difficulty of counting the number of CTCs accurately.
    This study aims towards a gentler diagnostic procedure so as not to lose too many CTCs during the sample preparation process and keep the cell’s viability.
    Furthermore, cells can form monolayer self-assembly after finishing immunofluorescence staining in order to avoid signal overlapped and to preserve the precious information of clinical morphology. Hence, a DS-SACA (Dynamic Staining Self-Assembly Cell Array) chip was designed in this study to perform gentle
    fluorescence-removal process by diffusion-based flow processes on CTCs and white blood cells (WBC) without centrifuging. This lead to a minimum manual handling of CTCs obtained in our study without any contamination. And the cells can form a monolayer for better view.

    目錄 第一章 緒論......................................................... 1 1.1 研究背景 ............................................................ 1 1.2 研究目標 ............................................................ 6 第二章 文獻回顧..................................................... 8 2.1 細胞篩選方法 ....................................................... 8 2.1.1 CellSearch 系統檢測循環腫瘤細胞 ................................. 8 2.1.2 免疫標定(Immuno-labeling) ..................................... 11 2.1.3 免疫磁球分離法(Immuno-magnetic separation) ..................... 13 2.1.4 免疫捕捉法(Immuno-capture) .................................... 15 2.1.5 免疫標定應用於臨床實驗 ........................................ 17 2.1.6 非免疫標定(Non-immunolabeling) ................................ 17 2.2 細胞自組裝陣列晶片(Self-Assemnly Cell Array Chip) ................. 21 2.2.1 自組裝陣列晶片之平台設計及原理 ................................ 21 2.2.2 平台製成及組裝 ................................................ 24 2.3 快速染色晶片3D-Dialysis chip .................................... 27 2.3.1 染色晶片結構設計 .............................................. 27 2.3.2 動態透析染色 .................................................. 27 2.4 接合 .............................................................. 28 第三章 實驗方法及設計.............................................. 30 3.1 實驗材料準備 ....................................................... 30 3.1.1 細胞培養 ...................................................... 30 3.1.2 器材使用 ...................................................... 32 3.1.3 藥品介紹 ...................................................... 33 3.2 晶片設計 .......................................................... 38 v 3.2.1 晶片側面三明治夾心結構 ........................................ 38 3.2.2 晶片外觀尺寸 .................................................. 39 3.2.3 晶片各部位計算 ................................................ 39 3.2.4 專利 …………………………………………………………………………….41 3.3 晶片工作原理 ....................................................... 42 3.3.1 動態透析染色 .................................................. 42 3.3.2 流速計算 ...................................................... 43 3.3.3 雷諾數計算 .................................................... 44 3.4 製程設計 .......................................................... 45 3.4.1 第一代DS-SACA 晶片 ............................................ 45 3.4.2 第二代DS-SACA 晶片 ............................................ 47 3.4.3 第三代DS-SACA 晶片 ............................................ 49 3.5 全血檢測步驟設計 .................................................. 51 3.6 細胞經離心機活性及損失率測試 ...................................... 52 3.7 材料螢光測試 ...................................................... 53 3.7.1 PDMS 孔洞狀薄膜螢光測試 ....................................... 53 3.7.2 SU8 孔洞狀薄膜螢光測試 ......................................... 53 3.8 PDMS 薄膜與SU8 薄膜應用於流場測試 .................................. 54 3.9 IRB 癌症病患檢體測試 ............................................... 55 3.9.1 檢體分離及染色流程 ............................................ 55 3.9.2 循環腫瘤細胞 CTC 影像辨識 ..................................... 57 3.10 晶片測試 .......................................................... 58 3.10.1 第二代DS-SACA 晶片 ........................................... 58 3.10.2 第三代DS-SACA 晶片 ............................................ 59 3.10.3 第二代與第三代比較 ............................................ 60 3.10.4 IRB 檢體於DS-SACA 晶片檢測結果 ................................ 60 第四章 實驗結果與討論............................................. 63 vi 4.1 細胞經離心機活性及損失率測試 ....................................... 63 4.2 材料螢光測試 .................................................... 64 4.2.1 PDMS 孔洞狀薄膜螢光測試 ....................................... 64 4.2.2 SU8 孔洞狀薄膜螢光測試 ......................................... 65 4.3 PDMS 薄膜與SU8 薄膜應用於流場測試 .................................. 66 4.4 晶片測試 ........................................................... 67 4.4.1 第二代DS-SACA 晶片 ............................................ 67 4.4.2 第三代DS-SACA 晶片 ............................................ 69 4.4.3 第二代與第三代之比較 .......................................... 70 4.4.4 IRB 檢體檢測結果 ............................................... 70 第五章 結論........................................................ 72 5 .1 後續展望 ........................................................ 72 第六章 參考文獻................................................... 75

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