跳到主要內容

臺灣博碩士論文加值系統

(3.229.137.68) 您好!臺灣時間:2021/07/25 18:19
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:沈耀安
研究生(外文):Yao-An Shen
論文名稱:鼻咽癌腫瘤幹細胞之篩選與鑑定
論文名稱(外文):Isolation and Identification of Cancer Stem Cell in Nasopharyngeal Carcinoma
指導教授:陳燕彰陳燕彰引用關係
指導教授(外文):Yann-Jang Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:124
中文關鍵詞:腫瘤幹細胞鼻咽癌抗輻射線抗藥性轉移微陣列
外文關鍵詞:cancer stem cellnasopharyngeal carcinomaCD44CD24side populationradioresistancetumor spheredrug resistancecDNA microarrayp63CK14metastasistumorigenicity
相關次數:
  • 被引用被引用:0
  • 點閱點閱:364
  • 評分評分:
  • 下載下載:62
  • 收藏至我的研究室書目清單書目收藏:0
腫瘤幹細胞為癌細胞中一群具有幹細胞特性的癌細胞,這些細胞能夠自我更新及導致臨床治療上的困難,如對治療產生抗性及造成轉移的發生。為了研究腫瘤幹細胞,我們可以透過亞細胞群(side population)分選法、辨認細胞表面抗原等方法來篩選腫瘤幹細胞。然而,鼻咽癌的腫瘤幹細胞表面抗原至今仍舊未知。
因此,我們利用腫瘤幹細胞本身之特性,像是抗藥性、抗輻射線以及能夠形成腫瘤球(tumor sphere),結合此三種特性自五株鼻咽癌細胞株(Tw01、Tw06、HONE-1、CNE-1、CNE-2)來篩選腫瘤幹細胞。當我們將這三種篩選方法合而為一,成為一個複合篩選法,便能更精準地尋找到腫瘤幹細胞。
藉此複合篩選法所篩選出來的腫瘤幹細胞,表現出高度的抗輻射線的能力、形成腫瘤球的數量、亞細胞群的比例、在軟洋菜膠(soft agar)的株落生長,以及轉移的潛力。這些候選的腫瘤幹細胞也表現高度的幹細胞基因、p63及CK14。有趣的是,這些外胚層鼻咽癌腫瘤幹細胞還能夠跨胚層分化為內胚層的肝細胞,他們甚至具有肝細胞的功能,我們也發現它們在分化時會改變細胞表面抗原。
我們接著在候選腫瘤幹細胞表面進行抗原掃讀,我們發現腫瘤幹細胞表面表現高量的CD44和CD24抗原。我們因此推論CD44和CD24雙陽性(double positive)的亞細胞群,是富含腫瘤幹細胞的。CD44和CD24雙陽性與CD44和CD24雙陰性比較,CD44和CD24雙陽性表現顯著的腫瘤幹細胞特性。五百顆CD44和CD24雙陽性細胞即能夠在NOD/SCID小鼠形成腫瘤,然而相同數量的CD44和CD24雙陰性細胞是無法形成腫瘤的。
再者,我們藉由轉染表皮間葉轉換因子Twist和山中因子Oct4、Sox2、Klf4、c-Myc得到鼻咽癌誘導腫瘤幹細胞。誘導腫瘤幹細胞在誘導後獲得腫瘤幹細胞的特性,並伴隨表現表面抗原CD44和CD24雙陽性的表型,這結果與我們先前所提出的鼻咽癌腫瘤幹細胞的表面富含CD44和CD24抗原吻合。
總括來說,複合篩選法能夠提供一個篩選腫瘤幹細胞的平台。CD44和CD24雙陽性能夠幫助我們找到腫瘤幹細胞,及設計針對鼻咽癌腫瘤幹細胞的藥物。p63和CK14具有相當的潛力當作鼻咽癌臨床預後和診斷之依據。未來,我們將繼續更深入且透徹地研究鼻咽癌腫瘤幹細胞的機制。
Cancer stem cell (CSC) describes the rare subset stem-like cancer cells that exist in tumors. These cancer cells are able to self-renew and cause clinical treatment obstacles, such as therapy resistance and metastasis. In order to investigate CSC, we isolate CSC through side population, cell surface markers, or other available methods. However, the surface marker of CSC in nasopharyngeal carcinoma (NPC) remains unknown.
Hence we took advantage of CSC’s natural properties, such as drug resistance, radioresistance, and the ability to form tumor spheres, to isolate CSC from five NPC cell lines (Tw01, Tw06, HONE-1, CNE-1, and CNE-2); the three isolation methods, when used together as one combination method, increased the precision in identifying CSC.
Selected from this isolation method, the CSC-candidates demonstrated significant radioresistance, tumor sphere formation, side population percentage, clonogenic growth in soft agar, and metastatic potential. These candidates also expressed high-level stemness genes, p63 and Cytokeratin 14. Interestingly, these ectoderm NPC cancer stem cell candidates were able to differentiate into endoderm liver cells, and they even possessed liver function. We also found the surface marker changed into another surface antigen during differentiation.
We then initiated surface marker screen on these CSC-candidates and located cell surface markers by identifying that CSCs have high level of CD44 and CD24 surface antigen expression. We inferred that CSC enriched subpopulation in NPC appeared as CD44+/CD24+. CD44+/CD24+ cells expressed significant CSC properties compared with differentiated cell-like CD44-/CD24- subpopulations. 500 CD44+/CD24+ cells formed tumor in NOD/SCID mice, while the same amount of CD44-/CD24- cells were not able to form tumor.
Furthermore, we generated NPC-induced cancer stem cell (iCSC) via transfecting Twist (EMT factor) as well as Oct4, Sox2, Klf4 and c-Myc (Yamanaka factors). iCSC acquired CSC properties along with CD44+/CD24+ antigen phenotype, which confirmed the CSC surface marker in NPC as we had predicted.
Taken together, the combination isolation method can serve as a model isolation method in CSC research. CD44+/CD24+ will help to identify CSC and aid in the subsequent drug design to target CSC in NPC. p63 and CK14 have prognosis and diagnosis marker potential. Looking ahead, we plan to investigate the mechanism of CSC in NPC even more thoroughly.
ACKNOWLEGEMENT 5
中文摘要 6
SUMMARY 8
INTRODUCTION 10
Nasopharyngeal Carcinoma 10
Definition 10
Symptoms 11
Risk factors 11
Treatment 13
Stem Cell 15
Properties 15
Cancer Stem Cell 16
Definition 16
Methodologies of Identifying Cancer Stem Cells 17
The Cancer Stem Cell Surface Markers 18
Induced Cancer Stem Cells (iCSC) 19
The Promising Combination Isolation Method of Selecting Cancer Stem Cell Clones with High Purity 20
RESULTS 22
Establishment of Cancer Stem Cell Clones 22
Radio-selection 22
Tumor sphere selection 23
Side population selection 23
Confirmation of Cancer Stem Cell Properties 24
Upregulation of stemness and EMT genes in cancer stem cell candidates 24
Detecting in vivo tumor formation ability of cancer stem cell candidates 25
Measuring the potency of cancer stem cell candidates 25
The NPC cancer stem cells changes the cell surface antigens when differentiated into liver cells 26
CD44/CD24 Double Positive Cells with Cancer Stem Cell Properties 27
NPC cancer stem cells express high level of CD44 and CD24 surface antigens 27
Upregulation of stemness and EMT genes in CD44+/CD24+ cells 28
Detecting in vivo tumor formation ability of CD44+/CD24+ cells 29
NPC iCSC expressed high level of CD44 and CD24 surface antigens 29
Upregulation of CK14 and p63 in NPC cancer stem cells 30
Comparison of Gene Expression among Parental Cells, Radioresistant Cells, and Cancer Stem Cells 31
Drug and Irradiation Treatment on Cancer Stem Cells in Nasopharyngeal Carcinoma 33
DISCUSSION 34
The Controversies of Cancer Stem Cell 34
Cancer Stem Cells from Combination Isolation Method and FACS Sorting by Surface Marker 35
The Structure of Tumor Spheres as Indicator of Cancer Stem Cell Clones Purity 37
The Naturally Existing Cancer Stem Cells and Induced Cancer Stem Cells 39
Probe into Metastasis Mechanism by Investigating the Potency of Cancer Stem Cell 40
The link among CK14, p63 and CD44 in NPC Cancer Stem Cells 42
Clinical Implication of Identifying Cancer Stem Cells Surface Markers 44
EXPEREMENTAL PROCEDURE 46
Cell Culture 46
Radio-Selection 46
Survival Fraction Assay 47
Tumor Sphere Selection 47
Side Population Selection 48
Cell Surface Marker Analysis 49
Real-time RT-PCR Analysis 49
Soft Agar Assay 50
MTT Cell proliferation Assay 50
Tumorigenesis Assay 51
REFERENCES 52
Brennan, B. (2006). Nasopharyngeal carcinoma. Orphanet J. of Rare Diseases 1, 23–28.
Humphrey, P.A., Dehner, L.P., and Pfeifer, J.D. (2008). The Washington manual of surgical pathology. Lippincott Williams & Wilkins, 39–41.
Hasselt, A.V., and Gibb, A.G. (1999). Nasopharyngeal carcinoma. The Chinese University Press, 105–111.
Hsu, M.M., and Tu, S.M. (1983). Nasopharyngeal carcinoma in Taiwan. Cancer 52, 362–368.
Old, L.J., Boyes, E.A., Oettgen, H.F., De Harven, E., Geering, G., Williamson, B., and Clifford, P. (1966). Precipitation antibody in human serum to an antigen present in cultured Burkitt’s lymphoma cells. Proc. Natl. Acad. Sci. USA 56, 1699–1704.
Miller, G., Tayler, N., Kolman, J., Baumann, R., Katz, D., Himmelfarb, H., Carrey, M., and Ptashne, M. (1990). How ZEBRA, a weak transactivator, exerts strong biological effect, in: Epstein-Barr virus and human disease. Humana Press, Clifton, New Jersey, 27.
Ernberg, I., Klein, G., Komilsby, F.M., Silvestre, D. (1974). Differentiation between early and late membrane antigen on human lymphoblastoid cell lines infected with Epstein-Barr virus. J. Natl. Cancer Inst. 53, 61–68.
Henle, G., Henle, and W., Klein, G. (1971). Demonstration of two distinct components in early antigen complex of Epstein-Barr virus-infected cells. Int. J. Cancer 8, 272–282.
Hummel, M., and Keiff, E. (1982). Mapping of polypeptides encoded by the Epstein-Barr virus genome in productive infection. Proc. Natl. Acad. Sci. USA 79, 5698–5702.
Teo P.M., Leung, S.F., Fowler, J., Leung, T.W., Tung, Y., O, S.K., Lee, W.Y., and Zee, B. (2000). Improved local control for early T-stage nasopharyngeal carcinoma - a tale of two Hospitals. Radiother. Oncol. 57, 155–166.
Cheng, S.H., Jian, J.J., and Tsai, S.Y. (2000). Long-term survival of nasopharyngeal carcinoma following concomitant radiotherapy and chemotherapy. Int. J. Radiat. Oncol. Biol. Phys. Biol. 48, 1323–1330.
Nishioka, T., Shirato, H., Arimoto, T., Masanori, K., and Toshihiro, K. (1997). Reduction of radiation-induced xerostomia in nasopharyngeal carcinoma using CT simulation with laser patient marking and three-field irradiation technique. Int. J. Radiat. Oncol. Biol. Phys. 38, 705–712.
Teo P.M.L. (2002). Intensity-modulated radiotherapy – a new standard for treating nasopharyngeal carcinoma? J. H.K. Coll. Radiol. 5, 211–212.
Chang, J.T., Ko, J.Y., and Hong, R.L. (2004). Recent advances in the treatment of nasopharyngeal carcinoma. J. Formos. Med. Assoc. 103, 496–510.
DeNittis, A.S., Liu, L., Rosenthal, D.I., and Machtay, M. (2004). Nasopharyngeal carcinoma treated with external radiotherapy, brachytherapy, and concurrent/adjuvant chemotherapy. Am. J. Clin. Oncol. 25, 93–5.
Nam, J., McLaughlin, J.K., and Blot, W.J. (1992). Cigarette smoking, alcohol and nasopharyngeal carcinoma: a case-control study among U.S. whites. J. Natl. Cancer Inst. 84, 619–622.
Mainou, B.A., and Raab-Traub, N. (2006). LMP1 strain variants: biological and molecular properties. J. Virol. 80, 6458–6468.
Ozyar, E., Ayhan, A., Korcum, A., and Atahan, I. (2004). Prognostic role of Epstein-Barr virus latent membrane protein-1 and interleukin-10 expression in patients with nasopharyngeal carcinoma. Cancer Invest. 22, 483–491.
Liu, L., Peng, J., Chang, H., and Hung, W. (2003). RECK is a target of Epstein-Barr virus latent membrane protein 1. Oncogene 22, 8263–8270.
Mei, Y.P., Zhou, J.M., Wang, Y., Huang, H., Deng, R., Feng, G.K., Zeng, Y.X., and Zhu, X.F. (2007). Silencing of LMP1 induces cell cycle arrest and enhances chemosensitivity through inhibition of AKT signaling pathway in EBVpositive nasopharyngeal carcinoma cells. Cell Cycle 6, 1379–1385.
Pegtel, D.M., Subramanian, A., Sheen, T.S., Tsai, C.H., Golub, T.R., and Thorley-Lawson, D.A. (2005). Epstein-Barr-virus-encoded LMP2A induces primary epithelial cell migration and invasion: possible role in nasopharyngeal carcinoma metastasis. J. Virol. 79, 15430–15442.
Yates, J.L., Warren, N., Sugden, B. (1985). Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 313, 812–815.
Wang, F., Tsang, S.F., Kurilla, M.G., Cohen, J.I., Kieff, E. (1990). Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1. J. Virol. 64, 3407–3416.
Ho, J.H.C. (1972). Nasopharyngeal carcinoma (NPC). Adv. Cancer Res. 15, 547–582.
Tam, J.S., and Murray, H.G.S. (1990). Nasopharyngeal carcinoma and Epstein-Barr virus-associated serologic markers. Ear. Nose Throat J. 69, 261–267.
Tsao, S.Y., and Chua, E.T. (1991). Current problems in radiotherapy, chemotherapy and staging of nasopharyngeal carcinoma (NPC). Ann. Acad. Med Singapore 20, 649–655.
Tsau, S.Y., and Shiu, W.C. (1990). Radiotherapy and chemotherapy for nasopharyngeal carcinoma. Ear. Nose Throat J. 69, 272–278.
Cheng, S.H., Jian, J.J., Tsai, S.Y., Yen, K.L., Chu, N.M., Chan, K.Y., Tan, T.D., Cheng, J.C., Leu, S.Y., Hsieh, C.Y., and Huang, A.T. (2000). Long-term survival of nasopharyngeal carcinoma following concomitant radiotherapy and chemotherapy. Int. J. Radiat. Oncol. Biol. Phys. 48, 1323–1330.
Yamashita, S., Kondo, M., and Hashimoto, S. (1985). Squamous cell carcinoma of the nasopharynx. An analysis of failure patterns after radiation therapy. Acta. Radiol. Oncol. 24, 315–320.
Chang, J.T.C., Chan, S.H., Lin, C.Y., Lin, T.Y., Wang, H.M., Liao, C.T., Wang, T.H., Lee, L.Y., and Cheng, A.J. (2007). Differentially expressed genes in radioresistant nasopharyngeal cancer cells: gp96 and GDF15. Mol. Cancer Ther. 6, 2271–2280.
Schöler, H.R. (2004). The Potential of Stem Cells: An Inventory. Humanbiotechnology as Social Challenge 47, 565-577
Zhang, X., Komaki, R., Wang, Li., Fang, B., and Chang, J.Y. (2008). Treatment of radioresistant stem-like esophageal cancer cells by an apoptotic gene-armed, telomerase-specific oncolytic adenovirus. Clin. Cancer Res. 14, 2813–2823.
Zhang, Q.B., Ji, X.Y., Huang, Q., Dong, J., Zhu, Y.D. and Lan, Q. (2006). Differentiation profile of brain tumor stem cells: a comparative study with neural stem cells. Cell Res. 16, 909–915.
Wang, J., Guo, L.P., Chen, L.Z., Zeng, Y.X., and Lu, S.H. (2007). Identification of cancer stem cell-like side population cells in human nasopharyngeal carcinoma cell line. Cancer Res. 67, 3716–3724.
Prince, M., Kaczorowski, R.S.A., Wolf, G.T., Kaplan, M.J., Dalerba, P., Weissman, I.L., Clarke, M.F., and Ailles, L.E. (2007). Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. PNAS 104, 973–978.
Chan, S.Y.Y., Choy, K.W., Tsao, S.W., Tao, Q., Tang, T., Chung, G.T.Y., and Lo, K.W. (2008). Authentication of nasopharyngeal carcinoma tumor lines. Int. J. Cancer 122, 2169–2171.
Li, C., Heidt, D.G., Dalerba, P., Burant, C.F., Zhang, L., Adsay, V., Wicha, M., Clarke, M.F., and Simeone, D.M. (2007). Identification of Pancreatic Cancer Stem Cells. Cancer Res. 67, 1030–1037.
Liu, J., and Johnston, M.R. (2002). Animal models for studying lung cancer and evaluating novel intervention strategies. Surg. Oncol. 11, 217–227.
Bibby, M.C. (2004). Orthotopic models of cancer for preclinical drug evaluation: advantages and disadvantages. Eur. J. Cancer 40, 852–857. Sun, B., Chen, M., Hawks, C., Hornsby, P.J., and Wang, X. (2006). Tumorigenic study on hepatocytes coexpressing SV40 with Ras. Mol. Carcinogenesis 45, 213–219.
Mani, S.A., Guo, W., Liao, M.J., Eaton, E.N., Ayyanan, A., Zhou, A.Y., Brooks, M., Reinhard, F., Zhang, C.C., Shipitsin, M., Campbell, L.L., Polyak, K., Brisken, C., Yang, J., and Weinberg, R.A. (2008). The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133, 704–715.
Senoo, M., Pinto, F., Crum, C.P., and McKeon, F. (2007). p63 Is essential for the proliferative potential of stem cells in Stratified Epithelia. Cell 129, 523–536.
Blanpain, C., and Fuchs, E. (2007). p63: revving up epithelial stem-cell potential. Nat. Cell Biol. 9, 731–733.
Kuo, P.L., Chiang, L.C., and Lin, C.C. (2002). Resveratrol-induced apoptosis is mediated by p53-dependent pathway in Hep G2 cells. Life Sci. 72, 23–34.
Atten, M.J., Godoy-Romero, E., Attar, B.M., Milson, T., Zopel, M., and Holian, O. (2005). Resveratrol regulates cellular PKC alpha and delta to inhibit growth and induce apoptosis in gastric cancer cells. Invest. New Drugs 23, 111–119.
Roccaro, A.M., Leleu, X., Sacco, A., Moreau, A.S., Hatjiharissi, E., Jia, X., Xu, L., Ciccarelli, B., Patterson, C.J., Ngo, H.T., Russo, D., Vacca, A., Dammacco, F., Anderson, K.C., Ghobrial, I.M., and Treon, S.P. (2008). Resveratrol exerts antiproliferative activity and induces apoptosis in Waldenstrom’s macroglobulinemia. Clin. Cancer Res. 14, 1849–1858.
Lu, K.H., Chen, Y.W., Tsai, P.H., Tsai, M.L., Lee, Y.Y., Chiang, C.Y., Kao, C.L., Chiou, S.H., Ku, H.H., Lin, C.H., and Chen, Y.J. (2009). Evaluation of radiotherapy effect in resveratrol-treated medulloblastoma cancer stem-like cells. Childs Nerv. Syst. 25, 543–550.
Amoh, Y., Kanoh, M., Niiyama, S., Hamada, Y., Kawahara, K., Sato, Y., Hoffman, R.M., and Katsuoka, K. (2009). Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral-nerve injury: An advantageous alternative to ES and iPS cells. J. Cell Biochem., in press.
Weinberg, R.A. (2007). The Biology of Cancer, cited in Basics: A mutinous group of cells on a greedy, destructive task, by Natalie Angier, New York Times.
Yang, A., Zhu, Z., Kapranov, P., McKeon, F., Church, G.M., Gingeras, T.R., and Struhl, K. (2006). Relationships between p63 binding, DNA sequence, transcription activity, and biological function in human cells. Mol. Cell 24, 593–602.
Flores, E.R. (2007). The roles of p63 in cancer. Cell Cycle 6, 300–304.
Senoo, M., Tsuchiya, I., Matsumura, Y., Mori, T., Saito, Y., Kato, H., Okamoto, T., Habu, S. (2001). Transcriptional dysregulation of the p73L/p63/p51/p40/KET gene in human squamous cell carcinomas: expression of DNp73L, a novel dominant-negative isoform, and loss of expression of the potential tumour suppressor p51. Br. J. Cancer 84, 1235–1241.
Foschini, M., Gaiba, A., Cocchi, R., Pennesi, M., Gatto, M., Frezza, G., Pession, A. (2004). Pattern of p63 expression in squamous cell carcinoma of the oral cavity. Virchows Archiv. 444, 332–339.
Rocco J.W., Leong C.O., Kuperwasser N., DeYoung M.P., Ellisen L.W. (2006). p63 mediates survival in squamous cell carcinoma by suppression of p73-dependent apoptosis. Cancer Cell 9, 45–56.
O’Guin, W.M., Galvin, S., Schermer, A., Sun, T.T. (1987). Patterns of keratin expression define distinct pathways of epithelial development and differentiation. Curr. Top Dev. Bio. 122, 97–125.
Coulombe, P.A., Kopan, R., Fuchs, E. (1989). Expression of keratin K14 in the epidermis and hair follicle: Insights into complex programs of differentiation. J. Cell Biol. 109, 2295–2312.
Fuchs E, Segre JA. (2000). Stem cells: A new lease on life. Cell 100:143–155.
Candi, E., Dinsdale, D., Rufini, A., Salomoni, P., Knight, R.A., Mueller, M., Krammer, P.H., and Melino, G. (2007). TAp63 and ΔNp63 in Cancer and Epidermal Development. Cell Cycle 6, 274–285.
Carroll, D.K., Carroll, J.S., Leong, C.O., Cheng, F., Brown, M., Mills, A.A., Brugge, J.S., and Ellisen, L.W. (2006). p63 regulates an adhesion programme and cell survival in epithelial cells. Nat. Cell Biol. 8, 551–561.
Boldrup, L., Coates, P.J., Gu, X., and Nylander, K. (2007). DeltaNp63 isoforms regulate CD44 and keratins 4, 6, 14 and 19 in squamous cell carcinoma of head and neck. J. Pathol. 213, 384–391.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊