利用膜過濾法分離卵巢癌細胞建立癌幹細胞系

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蘇翊碩(Yi-Shuo SU) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

利用膜過濾法分離卵巢癌細胞建立癌幹細胞系
(Isolation of Ovarian Cancer Cells to Establish Cancer Stem Cells Using Membrane Filtration Method)

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至系統瀏覽論文 (2026-8-20以後開放)

摘要(中)

卵巢癌是女性生殖系統中最致命的惡性腫瘤之一，其發病率和死亡率一直引起全球範圍內的關注。其中，癌症幹細胞是一種在腫瘤中具有幹細胞特性的細胞，它們具有自我更新和分化成多種細胞型的能力。這些幹細胞被認為在癌症的發展和轉移過程中扮演著關鍵角色。因此，針對癌症幹細胞的治療策略可能有助於阻止腫瘤的再生和轉移，從而提高治療效果和預後。
在這樣的背景下，本研究開發了一種膜過濾法。ES-2細胞株透過此方法，使用了不同的薄膜，分別是耐倫薄膜、聚乳酸-聚乙二醇酸薄膜和使用聚-L-賴氨酸與PVA-IA改質表面特性的的聚乳酸-聚乙二醇酸薄膜，透過改變薄膜的孔隙度度、厚度與表面性質達到純化CSCs的目的。純化效率將通過不同的檢測方式（1）流式細胞儀檢測CD44 & CD133的表達(2) 集落形成單位試驗。最後可以發現，在不同檢測方式的驗證下，透過與回收液和滲透液中的細胞相比，CSCs在遷移的細胞中具有更高的純度。
接下來，為了測試膜過濾法將癌細胞從腫瘤細胞群分離的可行性，我們將卵巢癌細胞ES-2與人類成纖維細胞 CG1639共培養進行模擬。分別使用紅色與藍色的細胞追蹤劑染色來進行標記，以便利用流式細胞儀及螢光顯微鏡進行鑑定。結果顯示，在使用癌症幹細胞純化效率最高的10%聚乳酸-聚乙二醇酸薄膜時，膜遷移的ES-2細胞的純度有明顯的提升，表明薄膜上有更高比例的癌細胞殘留。
這項技術顯示了作為分離癌症幹細胞（CSC）的可行策略的潛力，在即將到來的研究努力中具有精確醫學篩查的潛在應用。

摘要(英)

Ovarian cancer is one of the most malignant tumors in the female reproductive system, and its incidence and mortality rate have been a global concern. Cancer stem cells (CSCs) are a type of cells in tumors that possess stem cell characteristics, with the ability to self-renew and generate tumors in organs or tissues in different sites. These stem cells are believed to play a crucial role in the development and metastasis of cancer. Therefore, treatment strategies targeting cancer stem cells may help preventing tumor regeneration and metastasis, thereby improving treatment outcomes and prognosis. A membrane filtration method was used to purify cancer stem cells using ovarian cancer cells in this study. ES-2 ovarian cancer cell line was used to be purified by membrane filtration method via several membranes including Nylon 11 membranes, poly(lactide-co-glycolic acid)/silk screen (PLGA/SK) membranes, and PLGA/SK membranes modified with poly-L-lysine or polyvinylalcohol-co-itaconic acid (PVA-IA) to achieve purification of CSCs by changing the porosity, thickness, and surface charges of the membranes. The purification efficiency was assessed using the following methods: (1) flow cytometry to detect the expression of CD44 & CD133 (CSC markers), and (2) colony-forming unit assay. The migrating cells after permeation through the membranes were found to have higher purity of CSCs compared to the cells in the recovery and permeate solutions. In order to evaluate the feasibility of separation of cancer cells from a tumor cell population using membrane filtration, ovarian cancer cells ES-2 were mixed with human fibroblast cells CG1639 to generate the simulated solution of primary tumor cell solution. The cell staining with Cell Tracker Red and Blue were used to identify ovarian cancer cells and fibroblasts, respectively using flow cytometry assay and fluorescence microscopy evaluation. The results showed that the purity of migrated ES-2 cells from 10% PLGA/SK membranes was the highest, indicating a high proportion of cancer cells remained on the membranes after the permeation of mixed solution of ovarian cancer cells and fibroblasts.

關鍵字(中)

★ 卵巢癌
★ 癌症幹細胞
★ 尼龍膜
★ 膜過濾法
★ 聚乳酸-羥基乙酸共聚物/絲網
★ 膜改質

關鍵字(英)

★ Ovarian carcinoma
★ cancer stem cells
★ nylon membrane
★ membrane filtration method
★ poly(lactide-co-glycolic acid)/silk screen.
★ membrane modification

論文目次

Abstract I
摘要 III
Index of content V
Index of Figures VIII
Index of Tables XII
Introduction 1
1-1 Stem cells and cancer stem cells 1
1-1-1 Stem cells 1
1-1-1-1 Totipotent stem cells 2
1-1-1-2 Pluripotent stem cells 2
1-1-1-3 Multipotent stem cells 3
1-1-1-4 Unipotent stem cells 4
1-1-2 Cancer stem cells 5
1-2 Interaction of cancer cells with the microenvironment 6
1-2-1 Cancer-associated fibroblasts (CAFs) 6
1-2-2 Extracellular matrix (ECM) 8
1-2-3 Tumor-associated macrophages (TAMs) 9
1-3 Isolation process of cancer cells 11
1-3-1 Fluorescence-activated cell sorting (FACS) 11
1-3-2 Density gradient centrifugation 12
1-3-3 Cell surface marker-based separation (CSMBS) 13
1-3-4 Membrane filtration method 14
1-4 Identification of cancer stem cells 16
1-4-1 Expression of surface markers on cancer stem cells 16
1-4-2 Identification of carcinoembryonic antigen (CEA) through enzyme-linked immunosorbent assay (ELISA) technique 17
1-4-3 Colony formation assay (CFA) 19
1-4-4 Immunofluorescence staining (IF) 20
1-5 Goal of this study 22
Chapter 2 Materials and methods 23
2-1 Experimental materials 23
2-1-1 Cell sources 23
2-1-2 Membranes 23
2-1-3 Cell cultivation dishes 25
2-1-4 Phosphate buffered saline solution (PBS) 25
2-1-5 Cell culture medium 25
2-1-6 Evaluation of cancer stem cells 25
2-1-7CellTracker staining 26
2-2 Cell culture methods 26
2-2-1 Preparation of cell culture medium 26
2-2-2 Preparation of other experimental solutions 27
2-2-3 Cell cultivation 28
2-2-4 Passage of cell lines 28
2-2-5 Cell density measurement 29
2-2-6 Coculture method of cells and CellTracker staining of the cells 30
2-2-7 Preparation of PLGA-silk screen (PLGA/SK) membranes 32
2-2-8 Surface electrical modification of PLGA-silk screen membranes 33
2-3 Cell sorting method 34
2-4 Identification of cancer stem cells 36
2-4-1 Surface marker analysis of cancer stem cells 36
2-4-2 Soft agar colony formation assay 37
2-5 Characterization of membranes and cells 39
2-5-1 Scanning Electron Microscope (SEM) measurements 39
2-5-2 Contact angle 40
2-5-3 Zeta potential measurements 40
2-5-4 Dynamic light scattering ___DLS 40
Chapter 3 Results and discussion 42
3-1 Characterization of membranes 42
3-1-1 Scanning Electron Microscope (SEM) measurements 43
Unmodified membranes (PLGA/SK : silk 170 mesh size) 45
3-1-2 Porosity measurements 47
3-1-3 Zeta potential measurements 48
3-1-4 Contact angle measurements 51
3-2Characterization of ovarian cancer cell lines 53
3-2-1 Morphology 53
3-2-2 Dynamic Light Scattering 54
3-3 Membrane filtration method through PLGA/SK membranes prepared with different concentration of PLGA 57
3-3-1 The distribution of the ES-2 cells after membrane filtration 57
3-3-2 Surface marker analysis of cancer stem cells by flow cytometry 60
3-3-3 Soft agar colony formation assay of cancer stem cells 65
3-3-4 Isolation of ovarian cancer cells through membranes from different ratio of mixed cell solution by membrane filtration method 67
3-4 Cells isolated through PLGA/SK membranes modified with PVA-IA or PLL 78
3-4-1The distribution of the ES-2 cells after membrane filtration of the cells 78
3-4-2 Surface marker analysis of cancer stem cells by flow cytometry 80
3-4-3 Soft agar colony formation assay of cancer cells 86
3-4-4 Isolation of ovarian cancer cells from different ratio of mixed cell solution by membrane filtration method 89
Chapter 4 Conclusion 99
Reference 102

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指導教授

樋口亞紺(Akon Higuchi)

審核日期

2024-7-25

推文