臺灣博碩士論文加值系統

惡性腫瘤組織裡包含著一部分的特定族群，稱作為癌症起始細胞或癌症幹細胞，其細胞具有幹細胞特性、卓越的自我恢復能力，並且為促成惡性腫瘤形成、轉移的主要關鍵。目前為止，有部分的研究宣稱發現大腸癌癌症幹細胞的細胞表面標記，如：CD29、CD44、ESA、CD166、CD24、Lgr5、CD133以及ALDH-1，其中以CD133最廣為人知。然而這些細胞表面標記並沒有確切的證據顯示能有效的表現並標記大腸癌癌症幹細胞，而目前唯一可信的辦法只有藉由注射樣本細胞至老鼠體內觀察其腫瘤生成情況來確定其研究結果。
而我們的實驗是利用經由抗癌藥物添加至細胞培養液裡，再經由一至二周的體外培養，所存活的殘留細胞認定為具有抗藥性的大腸癌癌症細胞，並且我們相信這些存活細胞有極大可能是大腸癌癌症幹細胞，利用這些樣本細胞進行一連串的癌症幹細胞表現特性實驗，如：癌胚抗原表現量，CD133細胞表面標記表現以及動物體內注射樣本細胞之腫瘤生成觀察。其實驗結果顯示，具有抗藥性之癌症細胞其癌胚抗原表現量為未治療之癌症細胞表現量的二至四倍；但在細胞表面標記上，具有抗藥性之癌症細胞其表現量卻遠低於未治療之癌症細胞。而在腫瘤生成觀察上，具有抗藥性之癌症細胞在八周的觀察期後未有任何的腫瘤生成，但未治療之癌症細胞再注射的二到四周即有腫瘤生成。由以上實驗結果，我們推論具有抗藥性之癌症細胞能提高癌胚抗原的表現量，但並不是癌症幹細胞或癌症起始細胞。

Tumors contain a small subpopulation of cancer-initiating cells, known as cancer stem cells (CSCs) that exhibit a self-renewing capacity and are responsible for tumor generation. Cancer-initiating cells (CSCs) are the cells which would form tumors while having stem cell properties. It is suggested that CSCs persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Specific surface markers for colon CSCs have been reported, and CD133 is the most studied surface marker for colon CSCs. Several other colon CSC markers have been proposed; these include: ESA, CD44, CD166, Msi-1, CD29, CD24, Lgr5, and ALDH-1. However, exact and reliable surface markers of colon CSCs have not yet been identified rationally. The only reliable method for identifying and quantifying CSCs is the observation of tumor formation in a serial xenotransplantation model.
In this study, The drug-resistance cells of human colorectal adenocarcinoma tumor (LoVo) cells were found to produce more than two order higher amount of carcinoembryonic antigen (CEA) per cell, when less than 1% of the cells were survived in serum free medium or serum medium because of addition of anticancer drugs (5-FU, aspirin, oxaliplatin, cisplatin, Rosewell Park regime, or Mayo Clinic regime) in the culture medium. Drug-resistant LoVo cells were analyzed to determine whether those cells had CSC characteristics, e.g., small size of the cells/colonosphere and strong expression of CSC surface markers, as indicated by flow cytometry and immunohistochemistry analysis. Finally, in vivo tumorigenesis was examined by subcutaneously xenotransplanting the isolated drug-resistant LoVo cells into mice; we then evaluated whether the drug-resistant cells isolated in this study were CSCs. We found that drug-resistant cells, which comprised less than 1% of the LoVo human colon cancer cells that survived in serum-free or serum-containing medium supplemented with drugs (5-fluorouracil, acetylsalicylic acid, oxaliplatin, and cisplatin) were found to produce more than two orders higher than normal levels of carcinoembryonic antigen (CEA) per cell.
These results raised the question of whether CSCs could be isolated from drug-resistant colon cancer cells when anticancer drugs are added to the culture medium. The percentage of cells positive for CD133, which is known to be a typical marker of CSCs, decreased in parallel with a decrease in the cell survival rate after the addition of anticancer drugs in both the serum-free and serum-containing media. Drug-resistant LoVo cells had lower expression of CSC markers, including CD29, CD44, CD166, ALDH-1, Lgr5, and Msi-1, compared with the parental LoVo cells based on immunohistochemical examination.
It was concluded that the drug resistance cells of colon cancer cell line, which were isolated by addition of anticancer drugs in culture medium could enhance production of CEA in both serum free medium and serum medium, but were found not to be CSCs from tumor generation experiments in vivo, although CSCs were believed to be drug resistance cells in general.

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 Relationship between cancer cells and microenvironment 9
1-2 Analysis of CSCs by flow cytometry 13
1-3 CEA (carcinoembryonic antigen) detection by ELISA 15
1-4 Immunofluorescent staining (IF) 17
1-5 In vivo tumorigenic assay 19
Chapter 2 Materials and Methods 20
2-1 Cell lines 20
2-1-1 Cancer cell lines 20
2-2 Cell culture condition 20
2-2-1 LoVo cells 20
2-2-2 COLO205 cells 20
2-2-3 Defined serum-free condition 21
2-3 Preparation of chemotherapeutic agents 21
2-4 Preparation of buffer solution 22
2-5 In vitro chemotherapy 23
2-6 CEA production analysis 23
2-7 Flow cytometry 24
2-8 In vivo tumor challenge 25
2-9 Immunofluorescence 27
Chapter 3 Results and Discussion 28
3-1 The cell morphology of LoVo colon cancer cells and COLO205 colon cancer cells cultivated under drug treatment 28
3-2 The effect of drug-treatment on cell morphology 28
3-3 The CEA production of LoVo cells under anti-cancer drug treatment 38
3-4 Characterization of purified cancer stem cells (CSCs) 48
3-4-1 Putative cancer stem cell makers analyzed by immunofluorescence staining 48
3-5 In vivo tumorigenic bioassay 56
3-5-1 The tumorigenic potential of putative cancer stem cells sorted by chemotherapy 56
3-5-2 The tumorigenic potential of LoVo cells and COLO205 cells co-injected subcutaneously with several ECMs 56
Chapter 4 Conclusion 60
Supplementary data 62
Reference 66

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