臺灣博碩士論文加值系統

目前的醫學化療法可以消滅大部分癌細胞卻無法完全杜絕及防止癌症再復發而導致癌症病患的死亡率居高不下。這是由於在腫瘤中有一小群細胞具有生長，分化及自我更新的能力，稱為癌幹細胞，癌幹細胞具有很強的抗藥性。在全球統計上，大腸直腸癌位居第二大死因，且一半以上的病人在五年內死於其併發症。癌幹細胞被認為是腫瘤再復發的主要原因，因此癌幹細胞的純化及標定將有助於開發新的癌症診斷及治療法。然而目前為止並沒有專一性高且可信賴的癌幹細胞表面標定物可以標定癌幹細胞。
本研究利用人類大腸癌細胞株LoVo及大腸直腸癌病患的初期癌細胞為研究對象，藉由多種方法去純化，剔除及鑑定癌幹細胞。第一、利用無血清培養法純化癌幹細胞，第二、利用抗癌藥篩選癌幹細胞，第三、培養腫瘤細胞在生物材料 (pluronic immobilized plate) 上以純化或剔除癌幹細胞，第四、以傳統的磁珠分離法分離癌幹細胞，CD133和CD44目前較著名被假定為是癌幹細胞的表面標定物。最後以動物實驗法來驗證其致瘤性。癌幹細胞與多能性基因(Nanog, Oct4, Sox2)的關係也將被討論。
本研究結果顯示LoVo癌細胞在含有血清條件下培養10天比在無血清條件下培養10天更具有致瘤性，且LoVo癌細胞的致瘤性隨著培養時間增長而變強。細胞間的訊號傳遞被認為是致瘤性增強的主要原因，這假設我們命名為Class Leader Theory。癌幹細胞與細胞多能性並無直接關係。CD133+與CD133-皆可以在老鼠皮下形成腫瘤，而CD44+與CD44-皆不具致瘤力。我們認為藉由假定的腫瘤幹細胞標記物，亦即CD133及CD44，無法專一地標定腫瘤幹細胞，動物實驗法是目前唯一可以鑑定癌幹細胞的方法。
純化及標定腫瘤幹細胞一直以來都是很大的挑戰，然而在本研究中，藉由LoVo細胞培養在生醫材料(pluronic immobilized plate)上，我們成功的剔除LoVo癌細胞中絕大部分的癌幹細胞，這一發現將可在臨床應用上具有貢獻。

Patient always died of cancer after the failure of current therapies to eradicate the residue disease. It was thought that there is a subpopulation of cancer cells that has the ability of initiating the tumor relapse, termed tumor initiating cells or cancer stem cells. Colorectal carcinoma is the second leading cause of cancer death in the world. The caner stem cells (CSCs) appear able to fuel the growth of diverse tumors. The characterization of CSCs will help to devise novel diagnostic and therapy procedure. Cancer stem cells are seemed to be chemoresistant cells. However there are no validating cancer stem cell markers that can specifically identify the cancer stem cells until now.
The goal of this study is to purify, deplete and characterize the cancer stem cells in LoVo colon cancer cell lines and colon cancer patient specimens by several method such as cultivation under serum and serum-free conditions, isolation of the chemoresistant cells by anti-cancer drugs, and isolation by the culture on CDI-pluronic immobilized plates. The cells were compared to the cell isolated by traditional MACs-sorted method based on the putative surface markers of CSCs (i.e., CD44, and CD133). The tumorigenic ability was finally analyzed in in vivo bioassay. The pluripotent genes expression (i.e., Nanog, Oct4, and Sox2 ) through these conditioned cells were also discussed.
The results indicated that the LoVo cells cultivated under serum condition for 10 days have a higher tumorigenic ability than that cultivated for 1 day and that in the serum-free conditions. The cell-cell communication should be the major element to generate the tumor-initiation cells (cancer stem cells) among the tumor cells; this hypothesis is termed “class leader theory” in this study. There was no directly relationship between the cancer stem cells and the gene expression of pluripotency.
In this study, it was found that both CD133+ and CD133- cells could give rise to the
tumors in vivo whereas both CD44+ and CD44- could not generate the tumors in vivo. There is no validation of cancer stem cells markers that can specifically target at cancer stem cells. The only method to evaluate the cancer stem cells should be the direct in vivo bioassay.
To identify the cancer stem cells remains a big challenge. However, the most of the cancer stem cells were successively removed in LoVo colon cancer cells by cultivation of the LoVo cells on the pluronic immobilized plate. This could provide a great hope and implication in the clinical application.

摘要 I
Abstract II
誌謝 IV
Acknowledgments V
Table of Contents VI
List of Figures VIII
List of Tables XII
Chapter 1 Introduction 1
1-1 The relationship between stem cells and cancer stem cells 1
1-1-1 Stem cells 1
1-1-2 Cancers and Cancer stem cells 2
1-1-3 Identity of cancer stem cells 4
1-1-4 Implications of cancer stem cells 8
1-2 Analysis of CSCs by flow cytometry 10
1-3 Isolation of CSCs by magnetic-activated cell sorting (MACs) 12
1-4 Purification of stem on biomaterials having nano-segment 13
1-5 ELISA for CEA (carcinoembryonic antigen) 15
1-6 in vivo tumorigenic assay 17
1-7 Polymerase Chain Reaction (PCR) 17
1-7-1 Introduction of PCR 17
1-7-2 The procedure of PCR 18
1-7-3 Selection of primers 21
1-8 Immunofluorescent staining (IF) 23
1-9 Immunohistological staining (IHC) 24
Chapter 2 Materials and Methods 26
2-1 Cell lines and patient specimens 26
2-2 Cell culture condition 27
2-3 Preparation of chemotherapeutic agents 28
2-4 Preparation of buffer solution 29
2-5 Preparation of CDI activated pluronic Poly-L-lysine plate 29
2-6 X-ray photoelectron spectra (XPS) 30
2-7 Water contact angle 30
2-8 In vitro chemotherapy 31
2-9 CEA production analysis 31
2-10 Flow cytometry 33
2-11 MACs sorting method 33
2-12 Isolation of RNA and RT-PCR 35
2-12-1 Isolation of RNA 35
2-12-2 Single-strand cDNA synthesis 35
2-12-3 PCR (Polymerase Chain Reaction) steps 36
2-13 In vivo tumor challenge 38
2-14 Immunofluorescence 39
2-15 Immunohistochemistry 40
Chapter 3 Results and Discussion 42
3-1 Physical characteristics of CDI activated Pluronic poly-L-lysine plates 42
3-2 Morphology of LoVo colon cancer cells and primary cells cultivated under several conditions 47
3-2-1 The effect of serum on cell morphology 47
3-2-2 The effect of chemotherapy on cell morphology 48
3-2-3 The effect of the microenvironment on the cell morphology 58
3-3 The CEA production of LoVo cells under anti-cancer drug treatment 62
3-4 Characterization of purified cancer stem cells (CSC) 70
3-4-1 Putative cancer stem cell markers analyzed by flow cytometry 70
3-4-2 In vivo tumorigenic bioassay 76
3-4-3 Putative cancer stem cell markers and pluripotent genes analyzed by immunofluorescence staining
and RT-PCR 83
3-4-4 Pluripotent gene expression of purified cancer stem cells analyzed by RT-PCR 96
Chapter 4 Conclusion 100
Supplementary data 102
Reference 105

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