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研究生:江佩圜
研究生(外文):Chiang, Pei-Huan
論文名稱:利用螺旋式微流體高效率收集惡性肋膜積液中肺癌細胞
論文名稱(外文):Rapid Enrichment of Lung Cancer Cells from Malignant Pleural Effusion by Spiral Microfluidics
指導教授:李博仁李博仁引用關係
指導教授(外文):Li, Bor-Ran
口試委員:柯政昌李耀坤許鉦宗
口試日期:2019-12-27
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生醫工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:85
中文關鍵詞:螺旋式微流體惡性肋膜積液高通量連續性分離大小分離
外文關鍵詞:microfluidicsInertial forcelabel-freehigh-throughputPleural Effusion
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  • 被引用被引用:1
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肺癌是全球主要的死亡原因之一,根據2018年世界衛生組織統計,肺癌的死亡人數以176萬人居冠。其中,大約有85%的肺癌為非小細胞肺癌(NSCLC)。 NSCLC的早期症狀與感冒相似,容易被忽略,這就是大多數患者(約75%)在初次檢查時已經進入III期或IV期的原因之一。
癌症的確診需要取得病灶細胞,然而,肺癌患者在手術中的活組織相當罕見且不易取得,近期研究表明,在惡性肋膜積液(MPE)中可以發現大量肺癌細胞(LCC)。但是,肋膜積液體積龐大而且血球數量遠高於癌細胞數量。對於大量樣品而言,快速篩檢癌細胞以進行診斷非常重要。傳統上,樣品製備需要花費很多時間,而且在癌細胞數量稀少,血球數量極高的複雜背景情況之下,容易干擾癌細胞的檢測。因此,我們希望開發一種可以從MPE快速收集LCC並提高癌細胞純度的裝置,以進行進一步臨床和學術研究的新方法。
使用螺旋微流體裝置藉由流體動力學分離了癌細胞。這種無標記,高通量的設備可以連續地從大量肋膜積液中分離出癌細胞。由於曲線形微通道和出口設計,細胞受到慣性力(Inertial Lift Force)及迪恩力(Dean Force)的共同作用後,大多數血球細胞被過濾至出口O1至O3,同時癌細胞被聚焦到出口O4中。利用CD45和EpCAM抗體辨識白血球和癌細胞。螺旋式微流道裝置可以在6分鐘內快速收集癌細胞,與未經前處理的檢體相較之下提升10倍以上的純度,從而大大減少了樣品製備的時間並提高了診斷的準確性。
螺旋式微流體設備是非交叉污染,具有連續進樣,高通量,低成本且便於臨床樣品快速預處理等優點,是一個具有其他臨床使用及商業化潛力的裝置。
Lung cancer is one of the leading causes of death worldwide. In general, 85% of lung cancer are non-small cell lung cancer (NSCLC), accounting for the majority lung cancer cases. The early symptoms of NSCLC are mild and similar to cold that can be easily ignored. Such delay treatment causing about 75% of NSCLC patients are already facing in stage III or IV for their first observation.
The diagnosis of cancer requires the selection of cancer cells, but the complexity of the clinical biopsy (ex. blood and pleural effusion) would severely interfere the analysis of targeting cells. Additionally, compared with irrelevant molecules, the number of targeting cancer cells are extremely low. Therefore, it is important to screen out cancer cells from massive volume of biopsy in a short time.
In this study, we developed a spiral microfluidic device by fluid dynamics that can rapidly isolate the cancer cells, increasing their purity. This label-free and high-throughput device could continuously separate cancer cells and other irrelevant molecules from large-volume pleural effusion. Due to the curvilinear-shaped microchannel and the outlet design, most of the blood cells were flushed away to outlet 1 to 3, and the cancer cells were focused into outlet 4 through hydrodynamic force. The verification of WBCs and cancer cells are recognized by flow cytometry with CD45 and EpCAM antibodies. The cancer cells enrichment efficiency increased more than 10 times within 6 minutes.
The presented microfluidic device holds several advantages of non-cross-contaminated, low cost and high throughput with continuous injecting processing that facilitates the rapid pretreatment and accuracy of clinical diagnosis.
目錄
中文摘要 i
Abstract ii
誌謝 iii
目錄 iv
表目錄 vii
圖目錄 vii
第 一 章 緒論 1
1.1 全球癌症分布 1
1.2 肺癌診斷 4
1.3 肋膜積水簡介 4
1.3.1肋膜積水與癌症 5
1.3.2檢測肋膜積液 6
1.4 微流體與微機電系統簡介 7
1.4.1親和性分離 9
1.4.2物理性分離 11
1.5 基於慣性微流體分離大小不同粒子 13
1.5.1慣性聚焦現象 13
1.5.2流體基礎假設與公式 14
1.6 螺旋式微流道物理原理 15
1.6.1慣性升力 15
剪切梯度升力 15
壁面效應升力 16
1.6.2迪恩渦流 17
1.6.3側向位移 19
1.6.4限制比例 20
1.7 研究動機及目標 21
第 二 章 材料與方法 23
2.1 實驗材料 23
2.1.1實驗化學藥品 23
2.1.2生物性樣品 24
2.2 實驗儀器 25
2.3 實驗系統流程及架設 26
2.4 微流體晶片設計 28
2.5 微流體晶片製作 30
2.5.1精密雕刻與流道翻模 30
2.5.2微流道改質與封裝 31
2.6 細胞培養以及檢體處理 32
2.6.1細胞培養 33
2.6.2檢體前處理 33
2.7 細胞計數及影像分析 33
2.8 快速染色 34
2.9 免疫螢光染色 35
2.10 流式細胞儀分析 39
2.10.1流式細胞儀簡介 39
2.10.2高解析度螢光影像流式細胞儀 40
第 三 章 結果與討論 42
3.1 微流體設計 42
3.1.1晶片幾何設計 42
3.1.2螺旋式微流道中細胞分離機制 45
3.1.3螺旋式微流道參數探討 46
3.2 粒子模擬 50
3.3 細胞株模擬及驗證 51
3.3.1細胞株選擇 52
3.3.2細胞株大小統計 52
3.3.3細胞分離效果 53
3.4 細胞株混到檢體做模擬 56
3.4.1倒立式顯微鏡細胞影像圖 56
3.5 肋膜積液分離結果 57
3.5.1Imaging Flow Cytometry分析定義 57
3.5.2螺旋式流道的細胞集中效率 60
3.5.3各出口細胞濃度分布情形 61
3.5.4各出口肺癌細胞純度分布情形 64
3.5.5裝置比較 66
3.5.6檢體個別討論 68
第 四 章 結論與未來展望 73
4.1 結論 73
4.2 未來展望 75
參考文獻 76
附錄一 81
附錄二 83
流式細胞儀訊號分析 83
螢光影像流式細胞儀 85


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