抗阻塞微珠過濾裝置應用於大量收集血液中的循環腫瘤細胞

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姓名

吳承翰(Cheng-Han Wu) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

抗阻塞微珠過濾裝置應用於大量收集血液中的循環腫瘤細胞
(Anti-Clogging Hemofiltration Device with Zwitterionized Microspheres for Mass Collection of Circulating Tumor Cells)

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至系統瀏覽論文 (2025-1-16以後開放)

摘要(中)

循環腫瘤細胞在癌症臨床應用上被視為原位癌診斷的重要生物標記，隨著液態活檢與細胞分離技術的發展，循環腫瘤細胞已應用於腫瘤治療的即時監測以及精準醫療的預後與個人化的癌症治療。然而，極稀少的循環腫瘤細胞一直都是臨床應用發展受到限制的主因，儘管近年來循環腫瘤細胞分離技術已逐漸成熟，但仍以少量血液中分離檢測作為醫療診斷為主，尚無法真正地收集大量循環腫瘤細胞為癌症患者進行藥物篩選等個人化治療。因此，本研究建立一種血液過濾裝置，使用可避免血栓形成的一種雙離子材料，磺基甜菜鹼矽烷 (Sulfobetaine silane, SBSi)，修飾於二氧化矽微珠上並填充於管柱內，經由微珠間產生的空隙大小分離並收集大量血液中的循環腫瘤細胞，除了能夠降低癌症轉移的機率，還能夠大量收集循環腫瘤細胞提供後續診斷與個人化的治療。首先本研究用UV-Vis證實SBSi改質的微珠能有效地抵抗紅血球的貼附，接著對管柱過濾的各項變因進行測試，包含微珠尺寸，流速、填充床高度、管柱截面積以及沖提時間等，設計出對5×105個人類直腸癌細胞(HCT116)有著高達90%的移除率以及對血球細胞有著低滯留率的血液過濾條件，爾後利用細胞密度分離液將收集的HCT116細胞與微珠分離並測試密度分離法對細胞的回收率。另外也調查了長時間的過濾對HCT116細胞的生存率影響，以及成功地模擬了在高達2000毫升的循環過濾血量中收集並回收HCT116細胞，而大量收集的HCT116細胞能用於快速的細胞增殖。經由實驗結果證明了血液過濾裝置擁有大量移除並收集血液中的循環腫瘤細胞的能力，足以解決循環腫瘤細胞數量稀少的問題，具有將CTCs在癌症臨床的應用進一步發展並推向精準醫療的潛力。

摘要(英)

Clinical applications of circulating tumor cells (CTCs) as a cancer biomarker are considered as a useful index to provide valuable information, such as cancer detection, prognosis prediction, cancer drugs screening and monitoring the outcomes of systemic treatments. In recent decades, more and more studies developed reliable devices capturing and releasing CTCs from the blood of the cancer patients based on the physical and biological property of CTCs. However, the extremely rare CTCs still limited the development of precision medicine. Herein, the aim of the thesis is to develop a hemofiltration device by using silica microspheres packed in the column to collect a large number of CTCs from the blood. The silica microspheres were modified with sulfobetaine silane (SBSi) to endow anti-clotting property. The size of pores between microspheres was selected to selectively restrain CTCs which are larger than blood cells. In this study, we confirmed the antifouling of the red blood cells (RBCs) by UV-Vis, and examined various variables of column filtration, including the size of microspheres, flow rate, height of filter bed, column cross-section area, and elution times for observe the change of human colorectal cancer cells (HCT116) removal rate and blood cells retention rate, then designed the optimal condition to attain 90% removal rate with 5×105 HCT116 and low blood cells retention rate. The collected cancer cells were then harvested from the microspheres by using a cell density separation solution, and the cells recovery rate also had be investigated. Moreover, the cell viability had also be proven not affected by the flow rate we use. Finally, we demo 2000 mL blood with cancer cell flow through the hemofiltration device and successful recover the alive cancer cells. The experimental results showed that the hemofiltration device has the ability to collect mass cancer cells from the blood, and then the collected cells can be harvested and cultured in vitro. The potential of hemofiltration device in clinical application is expected to solve the problem that CTCs are extremely rare for development of precision medicine.

關鍵字(中)

★ 血液過濾裝置
★ 磺基甜菜鹼矽烷
★ 循環腫瘤細胞
★ 抗凝血

關鍵字(英)

★ hemofiltration
★ sulfobetaine silane
★ anti-clotting
★ circulating tumor cells

論文目次

中文摘要 i
Abstract ii
致謝辭 iv
目錄 v
圖目錄 ix
表目錄 xii
符號說明 xiii
第一章緒論 1
1-1 癌症轉移(Metastasis) 1
1-1-1 循環腫瘤細胞的臨床價值 1
1-1-2 循環腫瘤細胞團簇 2
1-1-3 循環腫瘤細胞的臨床應用 3
1-1-4 循環腫瘤細胞在臨床應用上的挑戰 4
1-2 循環腫瘤細胞分離技術 4
1-2-1 細胞物理特性與分離技術的應用與挑戰 5
1-2-2 細胞生物特性與分離技術的應用與挑戰 9
1-3 分離器材的應用與挑戰 12
1-3-1 凝血的機制 13
1-3-2 抗凝血的策略 14
1-4 表面改質 15
1-4-1 表面矽烷化(Silanization) 16
1-4-2 雙離子材料(Zwitterion) 16
1-4-3 磺基甜菜鹼矽烷(Sulfobetaine silane) 18
1-5 血液分離術(Apheresis) 18
1-5-1 雙離子材料應用於血液透析 18
第二章研究目的 20
第三章材料與方法 21
3-1 材料 21
3-2 實驗方法 22
3-2-1 SBSi的合成與鑑定 22
3-2-2 二氧化矽微珠表面改質與鑑定 23
3-2-3 管柱填充方法 24
3-2-4 HCT116細胞株的準備 25
3-2-5 細胞密度分離法 25
3-2-6 抗沾汙測試(Anti-fouling test) 26
3-2-7 流速之於細胞移除率 27
3-2-8 流速之於血球滯留率 29
3-2-9 填充床高度之於細胞移除率 30
3-2-10 管柱截面積之於細胞移除率 31
3-2-11 管柱截面積之於血球滯留率 32
3-2-12 沖提時間之於細胞移除率 33
3-2-13 細胞回收率 34
3-2-14 過濾時間之於細胞存活率 35
3-2-15 血液過濾裝置模擬 36
3-2-16 全血分離的HCT116細胞株增生 37
第四章結果與討論 38
4-1 鑑定SBSi (1H NMR) 38
4-2 鑑定SBSi對二氧化矽微珠的表面改質 (13C NMR) 39
4-3 SBSi改質的二氧化矽微珠抗紅血球貼附能力(Anti-fouling test) 40
4-4 管柱過濾的變因比較 41
4-4-1 鑑定微珠孔隙的大小 41
4-4-2 鑑定HCT116癌細胞大小 43
4-4-3 流速之於細胞移除率與血球滯留率 44
4-4-4 填充床高度之於細胞移除率 49
4-4-5 管柱截面積之於細胞移除率與血球滯留率 50
4-4-6 沖提時間之於細胞移除率 54
4-5 細胞回收率(Cells recovery rate) 56
4-6 過濾時間之於細胞存活率(Cells viability test) 58
4-7 血液過濾裝置的模擬 59
4-8 細胞增殖(Cells proliferation) 61
第五章結論 62
第六章未來工作 64
參考文獻 66
附錄1 73
常用公式 73
附錄2 74
填充床線性流速推導 74

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指導教授

黃俊仁(Chun-Jen Huang)

審核日期

2020-1-18

推文