使用耐綸薄膜以及PLGA薄膜過濾法來分離與建立專一性結腸癌病患單一細胞株

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汪佳樺(Jia-Hua Wang) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

使用耐綸薄膜以及PLGA薄膜過濾法來分離與建立專一性結腸癌病患單一細胞株
(Establishment of Patient-specific Cancer Cell Lines by Membrane Filtration Method via Nylon Mesh Filter and PLGA-Silk Screen Membranes)

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★ 羊水間葉幹細胞培養於接枝細胞外間質寡肽與環狀肽具有最佳表面硬度的生醫材料,其增殖能力及多能性之研究	★ 人類體細胞從組成誘導型多能性幹細胞培養在無飼養層上

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摘要(中)

根據世界衛生組織的數據顯示，癌症是全球前十大死因之一。癌症初始細胞又稱作癌症幹細胞，該細胞群被認為是造成腫瘤的發生、生長和轉移的重要因素，並且只佔腫瘤細胞群的一小部分。此外，癌症幹細胞需對細胞增生負重要責任，他們也比一般癌細胞對傳統化療更具有抵抗力。因此，病患專一性癌症細胞株的分離和建立變得極為重要。然而癌症幹細胞只佔腫瘤細胞群的1%–5%，也很難將癌症幹細胞與原代組織中的其他細胞區分開來。因此我們建立了一種膜過濾方法將這群癌症幹細胞純化，並同時從結腸癌組織中建立原代結腸癌細胞株。
膜過濾法使用了耐綸網膜以及聚乳酸羥基乙酸共聚物絲網膜，以達到分離以及純化這群癌症幹細胞的目的。我們透過數個步驟從結腸腫瘤組織中分離出原代結腸癌細胞，並以癌細胞懸浮液的方式，通過薄膜以進一步提高癌症幹細胞的比例。我們使用菌落形成檢驗法、表面標誌物檢驗法(CD44和CD133)以及癌胚胎抗原來測定癌症幹細胞和結腸癌細胞。市售的結腸癌細胞株也同樣在使用膜過濾法後被分析，以此作為原發性結腸癌組織溶液的模型。
使用膜過濾法來純化癌症幹細胞已分別在兩個結腸癌細胞株 LoVo 和 HT29上成功驗證，但卻顯示出不同的結果。透過流式細胞儀的檢測結果顯示，從膜遷移出來的LoVo細胞具有最高程度的癌症幹細胞標誌物表達。而這個結果再次被菌落形成法所驗證，從膜遷移出來的LoVo細胞對形成菌落具有很高的能力。然而，那些沒通過薄膜所蒐集到的HT29細胞，比其他部份具有更高的癌症幹細胞標誌物表達程度。而這部分的細胞在菌落形成檢驗法中，同樣顯示出很高的菌落形成能力。
我們期望透過膜過濾的方法，以通過具有不同孔徑、親和力和生物相容性的薄膜，來建立高純度的病患專一性癌症細胞株。該方法也被認為是一種具有前途的癌症幹細胞純化方法，並運用在未來發展精準且有效率的癌症治療上。

摘要(英)

Cancer is one of the top 10 leading cause of death in the world according to World Health Organization (WHO). Cancer-initiating cells (CICs), or cancer stem cells (CSCs), are primarily responsible for tumor initiation, growth, and metastasis and represent a few percentages of the total tumor cell population. Moreover, CSCs are strongly responsible for cancer proliferation and show stronger resistance than other cancer cells under conventional cancer therapy. Therefore, the isolation and establishment of patient-specific primary cancer cell line is extremely important. However, CSCs typically comprise 1%–5% of the total tumor cell population. It is difficult to distinguish CSCs from other cells in primary tissue. Therefore, a membrane filtration method is developed to target CSCs purification and establish primary colon cancer cell line from primary colon cancer tissue in this study. The membrane filtration method was used to isolate and purify CSCs via porous Nylon mesh filters or poly(lactic-co-glycolic acid)-silk screen membranes. The primary colon cancer cells were isolated from colon tumor tissues by several steps. The cancer cell solution was permeated through these membranes to further enhance CSCs proportion. CSCs and colon cancer cells were characterized using colony formation assay, surface marker (CD44 and CD133) assay, and expression of carcinoembryonic antigen using ELISA and flow cytometry assay. Colon cancer cell lines were also analyzed using the membrane filtration method as a model of primary colon cancer tissue solution. The purification method of CSCs using the membrane filtration method has been successfully verified using LoVo and HT29 colon cancer cell line but showed the different results. For LoVo cells, the cells that migrated from the membranes showed higher level expression of CSCs marker (CD133, CD44) analyzed using flow cytometry. This result was again confirmed by colony formation assay where the migrated cells from the membranes could generate high colony forming efficiency. For HT29 cells, the cells in the recovery solution had the highest CSCs marker expression. In addition, those cells in the recovery solution could also generate high colony forming efficiency after permeated through different membranes. It is expected that the high purity patient-specific colon cancer cell line could be established using the membrane filtration method via the membranes having different pore sizes, affinity and biocompatibility. This method is thought to be a promising approach for CSC purification, which can be applied for precision medicine screening in the future.

關鍵字(中)

★ 癌症幹細胞
★ 耐綸薄膜
★ 薄膜過濾法

關鍵字(英)

論文目次

Abstract I
摘要 III
Index of Contents IV
Index of Figure VIII
Index of Table XIII
Chapter 1 Introduction 1
1-1 Stem cells and cancer stem cells 1
1-1-1 Stem cells 1
1-1-2 Cancer stem cells 4
1-2 The effect of microenvironment on cancer stem cells 8
1-2-1 Cancer associated fibroblasts 8
1-2-2 Extracellular Matrix (ECM) 9
1-2-3 Substrate stiffness 10
1-3 Evaluation of cancer stem cells 12
1-3-1 Surface marker expression of cancer stem cells 12
1-3-2 Carcinoembryonic antigen (CEA) 13
1-3-3 Colony farmation asssay (CFA) 15
1-3-4 Immunofluorescence staining (IF) 17
1-4 Isolation process of cancer cells 19
1-4-1 Fluorescence-activated cell sorting (FACS) 19
1-4-2 Magnetic-activated cell sorting (MACS) 21
1-4-3 Membrane filtration method 23
1-5 The goal of this study 25
Chapter 2 Materials and methods 27
2-1 Experimental materials 27
2-1-1 Cell sources 27
2-1-2 Membranes 28
2-1-3 Cell cultivation dishes 28
2-1-4 Digestion and passage process 29
2-1-5 Ammonium-Chloride-Potassium (ACK) lysing buffer 29
2-1-6 Phosphate buffered saline solution (PBS) 29
2-1-7 Cell culture medium 29
2-1-8 Evaluation of cancer stem cells 30
2-2 Cell culture methods 31
2-2-1 Preparation of cell culture medium 31
2-2-2 Preparation of phosphate buffered saline solution (PBS) 31
2-2-3 Preparation of ACK lysis buffer 32
2-2-4 Preparation of collagenase digestion agent 32
2-2-5 Culture and passage of colon cancer cell lines 32
2-2-6 Cell density measurement 33
2-2-7 Extraction of primary colon cancer cells 34
2-2-8 Preparation of PLGA-silk screen membranes 36
2-3 Cell sorting methods 38
2-3-1 Membrane filtration method 38
2-3-2 Magnetic-activated cell sorting (MACS) method 40
2-4 Identification of cancer stem cells 42
2-4-1 Surface marker analysis of cancer stem cells 42
2-4-2 Evaluation of carcinoembryonic antigen concentration 42
2-4-3 In vitro tumorigenicity assay 44
2-5 Characterization of membranes 48
2-5-1 Scanning Electron Microscope (SEM) measurements 48
2-5-2 Primos Lite measurements 48
2-5-3 Zeta potential measurements 48
Chapter 3 Results and discussion 49
3-1 Characterization of membranes 49
3-1-1 Scanning Electron Microscope (SEM) measurements of the membranes 49
3-1-2 Primos Lite measurements 56
3-1-3 Zeta potential measurements 60
3-2 Characterization of colon cancer cell lines 65
3-2-1 Isolation of cancer stem cells from LoVo cell line by membrane filtration method 65
3-2-2 Isolation of cancer stem cells from HT29 cell line by membrane filtration method 68
3-2-3 Surface marker analysis of cancer stem cells by flow cytometry 72
3-2-4 Carcinoembryonic antigen concentration 89
3-2-5 Colony formation assay of colon cancer cells from each fraction 94
3-3 Characterization of primary cells 100
3-3-1 Extraction of cancer cells from colon cancer tissue 100
3-3-2 Carcinoembryonic antigen concentration of primary colon cancer cells 101
3-3-3 Colony formation assay (CFA) of primary colon cancer cells 102
3-3-4 Surface marker analysis of primary colon cancer cells using flow cytometry 103
3-3-5 Isolation and purification of primary colon cancer cells by membrane filtration method 104
3-3-6 Isolation and purification of primary colon cancer cells using MACS 108
Chapter 4 Conclusion 111
Reference 113
Appendix 125

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指導教授

樋口亞紺(Akon Higuchi)

審核日期

2021-8-11

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