臺灣博碩士論文加值系統

樹狀細胞(Dendritic Cell, DC)是目前已知功能最強的腫瘤抗原呈現細胞。實驗由成人週邊血中的單核球細胞(monocytes)分離出CD14+細胞，給予GM-CSF、IL-4生長因子以培養樹狀細胞。培養第五天給予TNF-α，利用流式細胞儀(Flow cytometry assay)分析其細胞表面標誌CD83，其表現在第六天逹最高58.6%，其它表面標誌如CD80、CD86、CD40、CD1a、HLA-DR,，其表現也逹50%以上，可推測CD14+細胞在此環境條件下可分化為樹狀細胞。利用酵素免疫吸附分析（enzyme-linked immunosorbent assay, ELISA）套組測定樹狀細胞分泌細胞素IL-12濃度，其IL-12濃度會隨著培養天數逐漸增加，由第三天49.83 pg/mL至第九天增加為556.5 pg/mL，由此可知實驗培養的樹狀細胞為成熟且具功能之樹狀細胞。此外，在培養第五天細胞表現FITC-Dextran強度最高，可推測培養中的樹狀細胞處於未成熟及成熟階段，推測此時可能為樹狀細胞捕捉及吞噬抗原最佳時間點，因此在實驗培養第五天給予5 μl/mL腫瘤抗原細胞角質素19或MCF-7腫瘤溶胞液，並與同源T細胞共同培養九天，經混合淋巴球反應(mixed lymphocyte reaction, MLR) 發現給予腫瘤抗原細胞角質素19，或MCF-7腫瘤溶胞液之樹狀細胞和T細胞共同培養72小時之後，其O.D.值相較於第0小時分別可增加1.2和1.17倍，144小時之後則可增加1.59和1.45 (p<0.05)，有顯著增加。共同培養之T細胞分泌細胞素INF-γ在給予腫瘤抗原細胞角質素19之樹狀細胞組為1992.15 pg/mL；MCF-7腫瘤溶胞液的樹狀細胞組為2187.61 pg/mL，可推測共同培養之T細胞正趨向分化。共同培養之T細胞對於有高表現細胞角質素19之MCF-7細胞株及不表現細胞角質素19之MES-SA細胞株毒殺能力分別為5.91 ± 2.76及1.33 ± 2.77 (p > 0.05)，沒有顯著差異。可能由於個體差異，或是腫瘤抗原角質素19之長度相較於抗原在細胞內經處理後成8-12個胺基酸過長，或是T細胞活化、增生能力之不足，或是抗原與T細胞之間human leucocyte antigen（HLA）typing未加考慮而影響。

Dendritic cells (DCs) are the most potent antigen-presenting cells (APC) within the immune system. Our study is to investigate an ex vivo model designed to test different strategies for the development of a therapeutic vaccine against cytokeratin 19 (CK19) overexpressed cancer based on CK19 fragment or MCF-7 (CK19 overexpressed cell line) tumor lysate pulsed DCs. We show here that highly purified CD14+ peripheral blood monocytes cultured with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) develop can differentiate into DCs with high efficacy (>95%). The phenotypes and surface marker of DCs are analyzed by flowcytometry. The mixed leukocyte responses are evaluated by water-soluble tetrazolium (WST-1) method for cell prolifereation assay. The cytokines of DCs and T cells are detected by enzyme-linked immunosorbent assay (ELISA). The T cells were generated by DCs pulsed with CK19 antigen or MCF-7 tumor lysate, and T cells cytotoxicity was measured by 51Cr release assay. The DCs derived from peripheral blood mononuclear cells expressed high levels of CD83, CD86, CD80, CD40, CD1a, HLA-DR and IL-12 at culture day 6. These cells displayed all phenotypic and morphologic characteristics of mature DCs and which are the most potent stimulatory cells in allogeneic mixed leukocyte reactions. In the coculture system of autologous T cells with activated DCs, the high level of interferon-γ(INF-γ) is detected in culture supernant. The cytotoxicity rate of cytotoxic T lymphocytes (CTL) induced by the Cytokeratin 19 tumor antigen-induced DCs was higher than that of CTL induced by MCF-7 tumor lysate. The data show that after treatment with CK19 antigen or MCF-7 tumor lysate, the DCs can stimulate T cell proliferation and induce cytotoxicity against MCF-7 cells. In our system, DCs and T cells culture platform are successfully established. To improve the cytotoxic effect of educated T cells on target cells, the dosage of tumor associated antigens may be the key issue.

中 文 摘 要 i
Abstrct iii
目 次 v
圖 次 vii
表 次 viii
第一章 緒 言 1
第二章 文獻探討 2
一、 正常免疫機制調控 2
二、 樹狀細胞 5
三、 樹狀細胞與癌症 7
第三章 材料與方法 11
一、 血球來源 11
二、 分離週邊血液單核球細胞 11
三、 分離CD14+細胞 11
四、 分離T細胞 12
五、 樹狀細胞培養 13
六、 T細胞培養 13
七、 樹狀細胞和T細胞共同培養方法 14
九、 腫瘤抗原細胞角質素19及MCF-7腫瘤溶胞液 15
十、 西方墨點法 15
十一、 流式細胞儀分析 17
十二、 樹狀細胞吞噬能力測定 17
十三、 混合淋巴球反應 18
十四、 細胞激素IL-10、IL-12和 INF-γ之分析 18
十五、 51Cr釋放法測定細胞毒殺反應 19
十六、 統計方法 20
第四章 結 果 22
一、 利用西方墨點法偵測細胞株的細胞角質素19蛋白質的表現 22
二、 誘導CD14+細胞分化為樹狀細胞其培養過程細胞形態上的變化 22
三、 誘導CD14+細胞分化為樹狀細胞培養過程細胞表面標誌變化 23
四、 誘導CD14+細胞分化為樹狀細胞培養過程細胞吞噬的能力 23
五、 誘導CD14+細胞分化為樹狀細胞培養過程細胞分泌細胞素IL-12之檢測 23
六、 給予腫瘤抗原細胞角質素19及MCF-7腫瘤溶胞液之樹狀細胞與同源T細胞共同培養過程中細胞形態上的變化 24
七、 T細胞增殖表現 24
八、 給予腫瘤抗原細胞角質素19及MCF-7腫瘤溶胞液之誘生樹狀細胞與T細胞共同培養之之後對於T細胞分泌細胞素INF-γ之分析 25
九、 不同的細胞毒殺性淋巴細胞對於不同的標的標的細胞之毒殺能力 26
第五章 討 論 44
參考文獻 50

白禮源等。 (1996). 甘龍醫用生理學。 台北， 軒圖書出版社。
麥麗敏等。 (2000). 簡明解剖生理學(第三版)。 台北，華杏。
Abbas, A.K., K.M. Murphy, and A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature 383:787-93.
Aiba, S. 1998. Maturation of dendritic cells induced by cytokines and haptens. Tohoku J Exp Med 184:159-72.
Albert, M.L., B. Sauter, and N. Bhardwaj. 1998. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392:86-9.
Azuma, M., M. Cayabyab, D. Buck, J.H. Phillips, and L.L. Lanier. 1992. CD28 interaction with B7 costimulates primary allogeneic proliferative responses and cytotoxicity mediated by small, resting T lymphocytes. J Exp Med 175:353-60.
Azuma, M., D. Ito, H. Yagita, K. Okumura, J.H. Phillips, L.L. Lanier, and C. Somoza. 1993. B70 antigen is a second ligand for CTLA-4 and CD28. Nature 366:76-9.
Banchereau, J., and R.M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245-52.
Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y.J. Liu, B. Pulendran, and K. Palucka. 2000. Immunobiology of dendritic cells. Annu Rev Immunol 18:767-811.
Bell, D., J.W. Young, and J. Banchereau. 1999. Dendritic cells. Adv Immunol 72:255-324.
Brossart, P., and M.J. Bevan. 1997. Presentation of exogenous protein antigens on major histocompatibility complex class I molecules by dendritic cells: pathway of presentation and regulation by cytokines. Blood 90:1594-9.
Cantrell, D.A., and K.A. Smith. 1984. The interleukin-2 T-cell system: a new cell growth model. Science 224:1312-6.
Caux, C., C. Dezutter-Dambuyant, D. Schmitt, and J. Banchereau. 1992. GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells. Nature 360:258-61.
Cella, M., F. Sallusto, and A. Lanzavecchia. 1997. Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol 9:10-6.
Cella, M., A. Engering, V. Pinet, J. Pieters, and A. Lanzavecchia. 1997. Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells. Nature 388:782-7.
Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, and G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J Exp Med 184:747-52.
Cheever, M.A., P.D. Greenberg, C. Irle, J.A. Thompson, D.L. Urdal, D.Y. Mochizuki, C.S. Henney, and S. Gillis. 1984. Interleukin 2 administered in vivo induces the growth of cultured T cells in vivo. J Immunol 132:2259-65.
Chou, P., and V. Chen. 1989. Mass screening for cervical cancer in Taiwan from 1974 to 1984. Cancer 64:962-8.
Corden, L.D., and W.H. McLean. 1996. Human keratin diseases: hereditary fragility of specific epithelial tissues. Exp Dermatol 5:297-307.
Coulombe, P.A., M.E. Hutton, R. Vassar, and E. Fuchs. 1991. A function for keratins and a common thread among different types of epidermolysis bullosa simplex diseases. J Cell Biol 115:1661-74.
Crawford, K., D. Gabuzda, V. Pantazopoulos, J. Xu, C. Clement, E. Reinherz, and C.A. Alper. 1999. Circulating CD2+ monocytes are dendritic cells. J Immunol 163:5920-8.
De Smedt, T., M. Van Mechelen, G. De Becker, J. Urbain, O. Leo, and M. Moser. 1997. Effect of interleukin-10 on dendritic cell maturation and function. Eur J Immunol 27:1229-35.
Dilioglou, S., J.M. Cruse, and R.E. Lewis. 2003. Function of CD80 and CD86 on monocyte- and stem cell-derived dendritic cells. Exp Mol Pathol 75:217-27.
Fields, R.C., K. Shimizu, and J.J. Mule. 1998. Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo. Proc Natl Acad Sci U S A 95:9482-7.
Fuchs, E., and D.W. Cleveland. 1998. A structural scaffolding of intermediate filaments in health and disease. Science 279:514-9.
Hart, D.N. 1997. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 90:3245-87.
Hsieh, C.S., S.E. Macatonia, C.S. Tripp, S.F. Wolf, A. O''Garra, and K.M. Murphy. 1993. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260:547-9.
Inaba, K., M. Pack, M. Inaba, H. Sakuta, F. Isdell, and R.M. Steinman. 1997. High levels of a major histocompatibility complex II-self peptide complex on dendritic cells from the T cell areas of lymph nodes. J Exp Med 186:665-72.
Kalinski, P., J.H. Schuitemaker, C.M. Hilkens, E.A. Wierenga, and M.L. Kapsenberg. 1999. Final maturation of dendritic cells is associated with impaired responsiveness to IFN-gamma and to bacterial IL-12 inducers: decreased ability of mature dendritic cells to produce IL-12 during the interaction with Th cells. J Immunol 162:3231-6.
Kasinrerk, W., T. Baumruker, O. Majdic, W. Knapp, and H. Stockinger. 1993. CD1 molecule expression on human monocytes induced by granulocyte-macrophage colony-stimulating factor. J Immunol 150:579-84.
Katz, S.I., K. Tamaki, and D.H. Sachs. 1979. Epidermal Langerhans cells are derived from cells originating in bone marrow. Nature 282:324-6.
Klein, E., S. Koch, B. Borm, J. Neumann, V. Herzog, N. Koch, and T. Bieber. 2005. CD83 localization in a recycling compartment of immature human monocyte-derived dendritic cells. Int Immunol 17:477-87.
Koch, F., U. Stanzl, P. Jennewein, K. Janke, C. Heufler, E. Kampgen, N. Romani, and G. Schuler. 1996. High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10. J Exp Med 184:741-6.
Kruse, M., O. Rosorius, F. Kratzer, D. Bevec, C. Kuhnt, A. Steinkasserer, G. Schuler, and J. Hauber. 2000. Inhibition of CD83 cell surface expression during dendritic cell maturation by interference with nuclear export of CD83 mRNA. J Exp Med 191:1581-90.
Langenkamp, A., M. Messi, A. Lanzavecchia, and F. Sallusto. 2000. Kinetics of dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells. Nat Immunol 1:311-6.
Larsson, M., J.F. Fonteneau, and N. Bhardwaj. 2001. Dendritic cells resurrect antigens from dead cells. Trends Immunol 22:141-8.
Lechmann, M., S. Berchtold, J. Hauber, and A. Steinkasserer. 2002. CD83 on dendritic cells: more than just a marker for maturation. Trends Immunol 23:273-5.
Macatonia, S.E., N.A. Hosken, M. Litton, P. Vieira, C.S. Hsieh, J.A. Culpepper, M. Wysocka, G. Trinchieri, K.M. Murphy, and A. O''Garra. 1995. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J Immunol 154:5071-9.
Maldonado-Lopez, R., T. De Smedt, P. Michel, J. Godfroid, B. Pajak, C. Heirman, K. Thielemans, O. Leo, J. Urbain, and M. Moser. 1999. CD8alpha+ and CD8alpha- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J Exp Med 189:587-92.
Markowicz, S., and E.G. Engleman. 1990. Granulocyte-macrophage colony-stimulating factor promotes differentiation and survival of human peripheral blood dendritic cells in vitro. J Clin Invest 85:955-61.
Moody, D.B., G.S. Besra, I.A. Wilson, and S.A. Porcelli. 1999. The molecular basis of CD1-mediated presentation of lipid antigens. Immunol Rev 172:285-96.
Moody, D.B., D.C. Young, T.Y. Cheng, J.P. Rosat, C. Roura-Mir, P.B. O''Connor, D.M. Zajonc, A. Walz, M.J. Miller, S.B. Levery, I.A. Wilson, C.E. Costello, and M.B. Brenner. 2004. T cell activation by lipopeptide antigens. Science 303:527-31.
Nakamura, I., K. Kajino, H. Bamba, F. Itoh, M. Takikita, and K. Ogasawara. 2004. Phenotypic stability of mature dendritic cells tuned by TLR or CD40 to control the efficiency of cytotoxic T cell priming. Microbiol Immunol 48:211-9.
Nestle, F.O., S. Alijagic, M. Gilliet, Y. Sun, S. Grabbe, R. Dummer, G. Burg, and D. Schadendorf. 1998. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 4:328-32.
Ng, H.T., C.C. Yuan, Y.Y. Kan, E.S. Ho, M.S. Yen, and K.C. Chao. 1995. An evaluation of chemotherapy in patients with cancer of the cervix and lymph node metastases. Arch Gynecol Obstet 256:1-4.
O''Doherty, U., R.M. Steinman, M. Peng, P.U. Cameron, S. Gezelter, I. Kopeloff, W.J. Swiggard, M. Pope, and N. Bhardwaj. 1993. Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium. J Exp Med 178:1067-76.
O''Garra, A. 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8:275-83.
P.F., M. 1999. Fundamental Immunology (4th edn) Elsevier Science.
Parronchi, P., S. Mohapatra, S. Sampognaro, L. Giannarini, U. Wahn, P. Chong, S. Mohapatra, E. Maggi, H. Renz, and S. Romagnani. 1996. Effects of interferon-alpha on cytokine profile, T cell receptor repertoire and peptide reactivity of human allergen-specific T cells. Eur J Immunol 26:697-703.
Porcelli, S., C.T. Morita, and M.B. Brenner. 1992. CD1b restricts the response of human CD4-8- T lymphocytes to a microbial antigen. Nature 360:593-7.
Quinlan, R.A., D.L. Schiller, M. Hatzfeld, T. Achtstatter, R. Moll, J.L. Jorcano, T.M. Magin, and W.W. Franke. 1985. Patterns of expression and organization of cytokeratin intermediate filaments. Ann N Y Acad Sci 455:282-306.
Rissoan, M.C., V. Soumelis, N. Kadowaki, G. Grouard, F. Briere, R. de Waal Malefyt, and Y.J. Liu. 1999. Reciprocal control of T helper cell and dendritic cell differentiation. Science 283:1183-6.
Rogge, L., L. Barberis-Maino, M. Biffi, N. Passini, D.H. Presky, U. Gubler, and F. Sinigaglia. 1997. Selective expression of an interleukin-12 receptor component by human T helper 1 cells. J Exp Med 185:825-31.
Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 179:1109-18.
Sallusto, F., C.R. Mackay, and A. Lanzavecchia. 2000. The role of chemokine receptors in primary, effector, and memory immune responses. Annu Rev Immunol 18:593-620.
Sallusto, F., M. Cella, C. Danieli, and A. Lanzavecchia. 1995. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med 182:389-400.
Santiago-Schwarz, F., N. Divaris, C. Kay, and S.E. Carsons. 1993. Mechanisms of tumor necrosis factor-granulocyte-macrophage colony-stimulating factor-induced dendritic cell development. Blood 82:3019-28.
Satthaporn, S., and O. Eremin. 2001. Dendritic cells (I): Biological functions. J R Coll Surg Edinb 46:9-19.
Schnurr, M., P. Galambos, C. Scholz, F. Then, M. Dauer, S. Endres, and A. Eigler. 2001. Tumor cell lysate-pulsed human dendritic cells induce a T-cell response against pancreatic carcinoma cells: an in vitro model for the assessment of tumor vaccines. Cancer Res 61:6445-50.
Shen, Z., G. Reznikoff, G. Dranoff, and K.L. Rock. 1997. Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules. J Immunol 158:2723-30.
Steinman, R.M. 1996. Dendritic cells and immune-based therapies. Exp Hematol 24:859-62.
Steinman, R.M., and Z.A. Cohn. 1973. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med 137:1142-62.
Stockwin, L.H., D. McGonagle, I.G. Martin, and G.E. Blair. 2000. Dendritic cells: immunological sentinels with a central role in health and disease. Immunol Cell Biol 78:91-102.
Tang, L.L., Z. Zhang, J.S. Zheng, J.F. Sheng, and K.Z. Liu. 2005. Phenotypic and functional characteristics of dendritic cells derived from human peripheral blood monocytes. J Zhejiang Univ Sci B 6:1176-81.
Thomas, R., L.S. Davis, and P.E. Lipsky. 1993. Isolation and characterization of human peripheral blood dendritic cells. J Immunol 150:821-34.
Trinchieri, G. 1995. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu Rev Immunol 13:251-76.
van der Pouw Kraan, T.C., L.C. Boeije, R.J. Smeenk, J. Wijdenes, and L.A. Aarden. 1995. Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production. J Exp Med 181:775-9.
van Kooten, C., and J. Banchereau. 1997. Functional role of CD40 and its ligand. Int Arch Allergy Immunol 113:393-9.
Verginis, P., and G. Carayanniotis. 2004. Experimental autoimmune thyroiditis (EAT) induced by the thyroglobulin peptide (2596-2608): influence of H-2 and non H-2 genes. Autoimmunity 37:529-33.
Vieira, P.L., P. Kalinski, E.A. Wierenga, M.L. Kapsenberg, and E.C. de Jong. 1998. Glucocorticoids inhibit bioactive IL-12p70 production by in vitro-generated human dendritic cells without affecting their T cell stimulatory potential. J Immunol 161:5245-51.
Vieira, P.L., E.C. de Jong, E.A. Wierenga, M.L. Kapsenberg, and P. Kalinski. 2000. Development of Th1-inducing capacity in myeloid dendritic cells requires environmental instruction. J Immunol 164:4507-12.
Wan, Y., and J. Bramson. 2001. Role of dendritic cell-derived cytokines in immune regulation. Curr Pharm Des 7:977-92.
Widmaier, R., Strang. 2003. Human Physiology McGraw-Hill Education, New York.
Winzler, C., P. Rovere, M. Rescigno, F. Granucci, G. Penna, L. Adorini, V.S. Zimmermann, J. Davoust, and P. Ricciardi-Castagnoli. 1997. Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J Exp Med 185:317-28.
Wright-Browne, V., K.L. McClain, M. Talpaz, N. Ordonez, and Z. Estrov. 1997. Physiology and pathophysiology of dendritic cells. Hum Pathol 28:563-79.
Yuan, C.C., L.C. Tsai, S.C. Hsu, H.T. Ng, S.J. Tsai, H.M. Chen, M.W. Hung, C.K. Ho, D.M. Ho, and T.J. Gill, 3rd. 1992. Production and characterisation of a monoclonal antibody (Cx-99) against cervical carcinoma. Br J Cancer 65:201-7.
Zitvogel, L., J.I. Mayordomo, T. Tjandrawan, A.B. DeLeo, M.R. Clarke, M.T. otze, and W.J. Storkus. 1996. Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines. J Exp Med 183:87-97.