利用膜過濾法從結腸癌組織中建立病患專一性細胞株

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姓名

黃偉倫(Wei-Lun Huang) 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

利用膜過濾法從結腸癌組織中建立病患專一性細胞株
(Establishment of Patient-Specific Cancer Cell Lines from Colon Cancer Tissues by Membrane Filtration Method)

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摘要(中)

癌症幹細胞 (CSCs, 癌症初始細胞) 通常佔總腫瘤細胞群的 1%-5%。而該細胞群被認為是影響腫瘤發生、生長以及轉移的主要原因。然而，在進行酶消化反應之後很難將癌細胞與原生組織中的其他細胞 (即纖維母細胞、血細胞等) 區分開。由於癌症幹細胞相較於其他細胞及普通腫瘤細胞，其具有較高的細胞遷移能力的特性。因此，我們開發了膜過濾及膜遷移的方法用來純化極少量的原代癌症幹細胞。並期望能透過膜過濾及膜遷移的方法從腫瘤組織中建立原代結腸癌細胞株，在臨床的應用上用於發展病患專一性的治療方法。
我們設計了一種膜過濾的方法，藉以從人類結腸癌組織中純化並進一步建立原代癌細胞株。本實驗中使用了孔徑 11 微米、20 微米的耐綸網膜以及不同聚乳酸羥基乙酸共聚物濃度3%, 5%, 和10%的聚乳酸羥基乙酸共聚物絲網膜作為膜過濾法中的材料。隨著聚乳酸羥基乙酸共聚物的濃度提升，所製備出的聚乳酸羥基乙酸共聚物絲網膜厚度也隨之增加。其中，聚乳酸羥基乙酸共聚物是具備生物可分解性及高生物相容性的一種高分子材料。而耐綸則是具備非常特殊的物理及化學性質，這些性質讓上述材料能夠更容易地應用在細胞培養的過程中。在原代癌細胞萃取的流程中，我們使用了第四型的膠原蛋白酶對結腸癌組織進行酶消化反應以取得癌細胞懸浮液。隨後，將消化後的結腸癌細胞懸浮液通過不同孔徑與不同生物相容性的薄膜並利用薄膜來捕捉原代的結腸癌細胞。我們預期當結腸癌細胞溶液透過膜過濾方式，並將膜經過一段時間的細胞培養後，原本留在膜上的細胞會逐步的遷移到細胞培養盤上 (此過程為膜遷移法)。之後，我們會使用流式細胞儀檢測各組份細胞對於結腸癌症幹細胞標誌物的表達程度。同時，我們利用軟性瓊脂糖菌落形成檢驗法來測定在經過膜過濾與膜遷移後純化的細胞，透過其形成菌落的能力並作為推估其惡性程度的指標。依我們目前的結果指出，從薄膜中遷移出來的細胞，其具有較高的癌症幹細胞標誌物的表達。同樣的，在軟性瓊脂糖菌落形成檢驗中，我們發現了在相同的條件下由薄膜中遷移出來的細胞其形成菌落的能力相較於其他組分來的高，且能夠形成較大的菌落。而本研究的最終目標是期望能夠利用膜過濾或者膜遷移的方式進一步的建立病患專一性癌症細胞株，並在未來應用在發展精準且有效率的癌症治療上。

摘要(英)

Cancer stem cells (CSCs, cancer-initiating cells) typically comprise 1%–5% of the total tumor cell population. This cell population is considered to be primarily responsible for tumor initiation, growth, and metastasis. However, it is hard to distinguish cancer cells from other cells (i.e. fibroblasts, blood cells, etc.) in primary tissue after enzyme digestion process. Therefore, We develop membrane filtration and migration method to target and purify rare primary CSCs based on their high migration mobility characteristics compared with other tissue cells. It is expected to establish the primary colon cancer cell line from primary tumor tissue by the membrane filtration and migration method for the development of patient specific therapy in clinical application.
We designed a membrane filtration method to purify and further establish primary colon cancer cell line from human colon cancer tissue. Nylon mesh filters with pore size of 11 μm and 20 μm and PLGA (poly (lactic-co-glycolic acid))-silk screen membranes with different PLGA concentration 3%, 5%, and 10% as the membranes are used in this study where PLGA has biodegradation and biocompatibility properties. With increasing PLGA concentration, the thickness of the PLGA-silk screen membranes also increased. Nylon has unique physical and chemical properties. These properties can also apply to cell culture process. In the primary cancer cell extraction procedures, the colon cancer tissue was digested by collagenase type IV at first. After digestion of the tissue, the primary colon cancer cell solution was permeated through the membranes with different pore size and different degree of biocompatibility. These membranes were used to capture primary colon cancer cells, and the cells on the membranes were expected to migrate into the culture dishes during the cultivation of the membranes in the culture medium (i.e., membrane migration method) after the filtration of colon cancer tissue solution through the membranes. Afterwards, the expression of cancer stem cell markers, CD133 and CD44 was evaluated by flow cytometry. Furthermore, the malignancy index of primary cells and the cells purified by the membrane filtration and migration method was evaluated by soft agarose colony forming assay. It is expected that the cells migrate from the membranes will have higher expression of the colon cancer stem cell surface markers. The similar results may be obtained in soft agarose colony forming assay where the cells show higher colony forming efficiency and bigger colony size. The goal of this project is to establish a patient-specific colon cancer cell line by the membrane filtration and/or migration method for precision medicine in the future.

關鍵字(中)

★ 結腸癌
★ 膜過濾
★ 病患專一性
★ 癌症細胞株

關鍵字(英)

★ colon cancer
★ membrane filtration
★ patient-specific
★ cancer cell lines

論文目次

Abstract I
摘要 V
Index of contents VII
Index of figure X
Index of table XIII
Chpater 1 Introduction 1
1-1 Stem cells and cancer stem cells 1
1-1-1 Stem cells 1
1-1-2 Cancer stem cells 3
1-2 Cells isolation methods 8
1-2-1 Fluorescence-activated cell sorting (FACS) 8
1-2-2 Magnetic-activated cell sorting (MACS) 10
1-2-3 Membrane filtration 14
1-2-4 Density gradient centrifugation 15
1-3 Identification of cancer stem cells 16
1-3-1 Cancer stem cell surface marker analysis 16
1-3-2 in vitro tumorigenicity assay 17
1-3-3 Immunofluorescence staining 19
1-3-4 Concentration detection of carcinoembryonic antigen 20
Chpater 2 Materials and methods 23
2-1 Experimental materials 23
2-1-1 Membrane 23
2-1-2 Cell sources 23
2-1-3 Cell cultivation dishes 24
2-1-4 Decellularization dish 24
2-1-5 Dissociation and passaging agent 25
2-1-6 Ammonium-Chloride-Potassium (ACK) lysing buffer 25
2-1-7 Phosphate buffer saline solution (PBS) 25
2-1-8 Culture medium 25
2-1-9 Identification of cancer stem cells 26
2-2 Experimental methods 26
2-2-1 PLGA-silk screen membrane 26
2-2-2 Preparation of the decellularization dishes 27
2-2-3 Cell culture medium 28
2-2-4 Preparation of the phosphate buffer saline solution (PBS) 29
2-2-5 Preparation of the digestion agent 29
2-2-6 Preparation of ACK lysis buffer 29
2-2-7 Cancer stem cells sorting method 30
2-2-8 Cell lines 32
2-2-9 Cell culture 34
2-2-10 Identification of cancer stem cells 36
Chpater 3 Results and discussion 41
3-1 Characterization of several membranes 41
3-2 Isolation of cancer stem cells from LoVo cell line by membrane filtration method 45
3-2-1 Characterization of the LoVo cells after membrane filtration method 45
3-2-2 Identification of the cancer stem cells from surface marker analysis using flow cytometry 49
3-2-3 Evaluation of tumorigenic potential of the cells from each fraction 54
3-3 Characterization of the primary cells 60
3-3-1 Isolation of the primary cancer cells by membrane filtration method 60
3-3-2 Modification of the isolation process of primary human colon cancer cells 62
3-3-3 The growth rate of primary human colon cancer cells 68
3-3-4 Flow cytometry analysis of primary human colon cancer cells 69
3-3-5 Purification of the primary human colon cancer cells by membrane filtration method 70
3-3-6 CEA concentration detection of primary human colon cancer cell 73
Chpater 4 Conclusion 77
Reference 79
Appendix 87

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指導教授

樋口亞紺(Akon Higuchi)

審核日期

2019-8-20

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