請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34853
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊雅雯 | |
dc.contributor.author | Yi-Fan Ma | en |
dc.contributor.author | 馬依帆 | zh_TW |
dc.date.accessioned | 2021-06-13T06:35:38Z | - |
dc.date.available | 2011-02-08 | |
dc.date.copyright | 2006-02-08 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-01-11 | |
dc.identifier.citation | 1. Podolsky,D.K. Mucosal immunity and inflammation. V. Innate mechanisms of mucosal defense and repair: the best offense is a good defense. Am. J. Physiol 277, G495-G499 (1999).
2. Aderem,A. & Underhill,D.M. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17, 593-623 (1999). 3. Svanborg,C., Godaly,G., & Hedlund,M. Cytokine responses during mucosal infections: role in disease pathogenesis and host defence. Curr. Opin. Microbiol. 2, 99-105 (1999). 4. Tomlinson,S. Complement defense mechanisms. Curr. Opin. Immunol. 5, 83-89 (1993). 5. Janeway,C.A., Jr. & Medzhitov,R. Innate immune recognition. Annu. Rev. Immunol. 20, 197-216 (2002). 6. Medzhitov,R. & Janeway,C.A., Jr. Innate immune recognition and control of adaptive immune responses. Semin. Immunol. 10, 351-353 (1998). 7. Estcourt,M.J., Ramsay,A.J., Brooks,A., Thomson,S.A., Medveckzy,C.J., & Ramshaw,I.A. Prime-boost immunization generates a high frequency, high-avidity CD8(+) cytotoxic T lymphocyte population. Int. Immunol. 14, 31-37 (2002). 8. Dutton,R.W., Bradley,L.M., & Swain,S.L. T cell memory. Annu. Rev. Immunol. 16, 201-223 (1998). 9. Zinkernagel,R.M., Bachmann,M.F., Kundig,T.M., Oehen,S., Pirchet,H., & Hengartner,H. On immunological memory. Annu. Rev. Immunol. 14, 333-367 (1996). 10. Ahmed,R. & Gray,D. Immunological memory and protective immunity: understanding their relation. Science 272, 54-60 (1996). 11. Mackay,C.R. Homing of naive, memory and effector lymphocytes. Curr. Opin. Immunol. 5, 423-427 (1993). 12. Rogers,P.R., Dubey,C., & Swain,S.L. Qualitative changes accompany memory T cell generation: faster, more effective responses at lower doses of antigen. J. Immunol. 164, 2338-2346 (2000). 13. Sallusto,F., Lenig,D., Forster,R., Lipp,M., & Lanzavecchia,A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708-712 (1999). 14. Banchereau,J. & Steinman,R.M. Dendritic cells and the control of immunity. Nature 392, 245-252 (1998). 15. Bukowski,J.F., Morita,C.T., & Brenner,M.B. Human gamma delta T cells recognize alkylamines derived from microbes, edible plants, and tea: implications for innate immunity. Immunity. 11, 57-65 (1999). 16. Modlin,R.L. & Sieling,P.A. Immunology. Now presenting: gammadelta T cells. Science 309, 252-253 (2005). 17. Jondal,M., Schirmbeck,R., & Reimann,J. MHC class I-restricted CTL responses to exogenous antigens. Immunity. 5, 295-302 (1996). 18. Constant,S.L. & Bottomly,K. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu. Rev. Immunol. 15, 297-322 (1997). 19. Janeway,C.A., Jr. A tale of two T cells. Immunity. 8, 391-394 (1998). 20. Lopez-Botet,M. & Bellon,T. Natural killer cell activation and inhibition by receptors for MHC class I. Curr. Opin. Immunol. 11, 301-307 (1999). 21. Parker,L.C., Whyte,M.K., Dower,S.K., & Sabroe,I. The expression and roles of Toll-like receptors in the biology of the human neutrophil. J. Leukoc. Biol. 77, 886-892 (2005). 22. Dombrowicz,D. & Capron,M. Eosinophils, allergy and parasites. Curr. Opin. Immunol. 13, 716-720 (2001). 23. Marone,G., Triggiani,M., & de Paulis,A. Mast cells and basophils: friends as well as foes in bronchial asthma? Trends Immunol. 26, 25-31 (2005). 24. Ardavin,C., Amigorena,S., & Reis e Sousa Dendritic cells: immunobiology and cancer immunotherapy. Immunity. 20, 17-23 (2004). 25. Sallusto,F., Cella,M., Danieli,C., & Lanzavecchia,A. 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 (1995). 26. Stewart,A.J. & Devlin,P.M. The history of the smallpox vaccine. J. Infect. (2005). 27. Kieny,M.P. & Girard,M.P. Human vaccine research and development: An overview. Vaccine 23, 5705-5707 (2005). 28. Poland,G.A. Acellular pertussis vaccines: new vaccines for an old disease. Lancet 347, 209-210 (1996). 29. Liu,M.A. Vaccine developments. Nat. Med. 4, 515-519 (1998). 30. Singh,M. & O'Hagan,D. Advances in vaccine adjuvants. Nat. Biotechnol. 17, 1075-1081 (1999). 31. Ada,G.L. The immunological principles of vaccination. Lancet 335, 523-526 (1990). 32. McDonnell,W.M. & Askari,F.K. DNA vaccines. N. Engl. J. Med. 334, 42-45 (1996). 33. Ulmer,J.B., Donnelly,J.J., Parker,S.E., Rhodes,G.H., Felgner,P.L., Dwarki,V.J., Gromkowski,S.H., Deck,R.R., DeWitt,C.M., Friedman,A., & . Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259, 1745-1749 (1993). 34. Fynan,E.F., Webster,R.G., Fuller,D.H., Haynes,J.R., Santoro,J.C., & Robinson,H.L. DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc. Natl. Acad. Sci. U. S. A 90, 11478-11482 (1993). 35. Krishnan,S., Haensler,J., & Meulien,P. Paving the way towards DNA vaccines. Nat. Med. 1, 521-522 (1995). 36. Marwick,C. Exciting potential of DNA vaccines explored. JAMA 273, 1403-1404 (1995). 37. Fynan,E.F., Webster,R.G., Fuller,D.H., Haynes,J.R., Santoro,J.C., & Robinson,H.L. DNA vaccines: a novel approach to immunization. Int. J. Immunopharmacol. 17, 79-83 (1995). 38. Selby,M., Walker,C.M., & Ulmer,J.B. Mechanisms of action of DNA vaccines. Expert. Opin. Investig. Drugs 7, 1987-1995 (1998). 39. Gurunathan,S., Klinman,D.M., & Seder,R.A. DNA vaccines: immunology, application, and optimization*. Annu. Rev. Immunol. 18, 927-974 (2000). 40. Sasaki,S., Takeshita,F., Xin,K.Q., Ishii,N., & Okuda,K. Adjuvant formulations and delivery systems for DNA vaccines. Methods 31, 243-254 (2003). 41. Kim,J.J., Yang,J.S., Montaner,L., Lee,D.J., Chalian,A.A., & Weiner,D.B. Coimmunization with IFN-gamma or IL-2, but not IL-13 or IL-4 cDNA can enhance Th1-type DNA vaccine-induced immune responses in vivo. J. Interferon Cytokine Res. 20, 311-319 (2000). 42. Kim,J.J., Yang,J.S., Dentchev,T., Dang,K., & Weiner,D.B. Chemokine gene adjuvants can modulate immune responses induced by DNA vaccines. J. Interferon Cytokine Res. 20, 487-498 (2000). 43. Kim,J.J., Nottingham,L.K., Wilson,D.M., Bagarazzi,M.L., Tsai,A., Morrison,L.D., Javadian,A., Chalian,A.A., Agadjanyan,M.G., & Weiner,D.B. Engineering DNA vaccines via co-delivery of co-stimulatory molecule genes. Vaccine 16, 1828-1835 (1998). 44. Kim,J.J., Bagarazzi,M.L., Trivedi,N., Hu,Y., Kazahaya,K., Wilson,D.M., Ciccarelli,R., Chattergoon,M.A., Dang,K., Mahalingam,S., Chalian,A.A., Agadjanyan,M.G., Boyer,J.D., Wang,B., & Weiner,D.B. Engineering of in vivo immune responses to DNA immunization via codelivery of costimulatory molecule genes. Nat. Biotechnol. 15, 641-646 (1997). 45. Sasaki,S., Takeshita,F., Oikawa,T., Kojima,Y., Xin,K.Q., Okuda,K., & Ishii,N. Improvement of DNA vaccine immunogenicity by a dual antigen expression system. Biochem. Biophys. Res. Commun. 315, 38-43 (2004). 46. Selby,M., Walker,C.M., & Ulmer,J.B. Mechanisms of action of DNA vaccines. Expert. Opin. Investig. Drugs 7, 1987-1995 (1998). 47. Fynan,E.F., Webster,R.G., Fuller,D.H., Haynes,J.R., Santoro,J.C., & Robinson,H.L. DNA vaccines: a novel approach to immunization. Int. J. Immunopharmacol. 17, 79-83 (1995). 48. Krishnan,S., Haensler,J., & Meulien,P. Paving the way towards DNA vaccines. Nat. Med. 1, 521-522 (1995). 49. Marwick,C. Exciting potential of DNA vaccines explored. JAMA 273, 1403-1404 (1995). 50. McDonnell,W.M. & Askari,F.K. DNA vaccines. N. Engl. J. Med. 334, 42-45 (1996). 51. Fynan,E.F., Webster,R.G., Fuller,D.H., Haynes,J.R., Santoro,J.C., & Robinson,H.L. DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc. Natl. Acad. Sci. U. S. A 90, 11478-11482 (1993). 52. Yang,N.S., Burkholder,J., Roberts,B., Martinell,B., & McCabe,D. In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proc. Natl. Acad. Sci. U. S. A 87, 9568-9572 (1990). 53. Daniels,G.A., Sanchez-Perez,L., Diaz,R.M., Kottke,T., Thompson,J., Lai,M., Gough,M., Karim,M., Bushell,A., Chong,H., Melcher,A., Harrington,K., & Vile,R.G. A simple method to cure established tumors by inflammatory killing of normal cells. Nat. Biotechnol. 22, 1125-1132 (2004). 54. Bonnotte,B., Gough,M., Phan,V., Ahmed,A., Chong,H., Martin,F., & Vile,R.G. Intradermal injection, as opposed to subcutaneous injection, enhances immunogenicity and suppresses tumorigenicity of tumor cells. Cancer Res. 63, 2145-2149 (2003). 55. Kutzler,M.A. & Weiner,D.B. Developing DNA vaccines that call to dendritic cells. J. Clin. Invest 114, 1241-1244 (2004). 56. Cui,Z., Han,S.J., Vangasseri,D.P., & Huang,L. Immunostimulation mechanism of LPD nanoparticle as a vaccine carrier. Mol. Pharm. 2, 22-28 (2005). 57. Chen,P.W., Wang,M., Bronte,V., Zhai,Y., Rosenberg,S.A., & Restifo,N.P. Therapeutic antitumor response after immunization with a recombinant adenovirus encoding a model tumor-associated antigen. J. Immunol. 156, 224-231 (1996). 58. Borysiewicz,L.K., Fiander,A., Nimako,M., Man,S., Wilkinson,G.W., Westmoreland,D., Evans,A.S., Adams,M., Stacey,S.N., Boursnell,M.E., Rutherford,E., Hickling,J.K., & Inglis,S.C. A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer. Lancet 347, 1523-1527 (1996). 59. Pardoll,D.M. Cancer vaccines. Nat. Med. 4, 525-531 (1998). 60. Hsiao,C.D., Hsieh,F.J., & Tsai,H.J. Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish. Dev. Dyn. 220, 323-336 (2001). 61. Fu,Y., Wang,Y., & Evans,S.M. Viral sequences enable efficient and tissue-specific expression of transgenes in Xenopus. Nat. Biotechnol. 16, 253-257 (1998). 62. Hoiseth,S.K. & Stocker,B.A. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238-239 (1981). 63. Pan,Z.K., Ikonomidis,G., Pardoll,D., & Paterson,Y. Regression of established tumors in mice mediated by the oral administration of a recombinant Listeria monocytogenes vaccine. Cancer Res. 55, 4776-4779 (1995). 64. Pan,Z.K., Ikonomidis,G., Lazenby,A., Pardoll,D., & Paterson,Y. A recombinant Listeria monocytogenes vaccine expressing a model tumour antigen protects mice against lethal tumour cell challenge and causes regression of established tumours. Nat. Med. 1, 471-477 (1995). 65. Singh,M. & O'Hagan,D. Advances in vaccine adjuvants. Nat. Biotechnol. 17, 1075-1081 (1999). 66. Mesa,C. & Fernandez,L.E. Challenges facing adjuvants for cancer immunotherapy. Immunol. Cell Biol. 82, 644-650 (2004). 67. Baylor,N.W., Egan,W., & Richman,P. Aluminum salts in vaccines--US perspective. Vaccine 20 Suppl 3, S18-S23 (2002). 68. Mesa,C. & Fernandez,L.E. Challenges facing adjuvants for cancer immunotherapy. Immunol. Cell Biol. 82, 644-650 (2004). 69. Lien,E. & Golenbock,D.T. Adjuvants and their signaling pathways: beyond TLRs. Nat. Immunol. 4, 1162-1164 (2003). 70. Takeda,K., Kaisho,T., & Akira,S. Toll-like receptors. Annu. Rev. Immunol. 21, 335-376 (2003). 71. Kaisho,T. & Akira,S. Toll-like receptors as adjuvant receptors. Biochim. Biophys. Acta 1589, 1-13 (2002). 72. Akira,S., Takeda,K., & Kaisho,T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2, 675-680 (2001). 73. Aderem,A. & Ulevitch,R.J. Toll-like receptors in the induction of the innate immune response. Nature 406, 782-787 (2000). 74. Medzhitov,R. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1, 135-145 (2001). 75. Akira,S. & Sato,S. Toll-like receptors and their signaling mechanisms. Scand. J. Infect. Dis. 35, 555-562 (2003). 76. Freytag,L.C. & Clements,J.D. Mucosal adjuvants. Vaccine 23, 1804-1813 (2005). 77. Sanders,M.T., Brown,L.E., Deliyannis,G., & Pearse,M.J. ISCOM-based vaccines: the second decade. Immunol. Cell Biol. 83, 119-128 (2005). 78. Zinkernagel,R.M., Ehl,S., Aichele,P., Oehen,S., Kundig,T., & Hengartner,H. Antigen localisation regulates immune responses in a dose- and time-dependent fashion: a geographical view of immune reactivity. Immunol. Rev. 156, 199-209 (1997). 79. Cao,Y., Toben,C., Na,S.Y., Stark,K., Nitschke,L., Peterson,A., Gold,R., Schimpl,A., & Hunig,T. Induction of experimental autoimmune encephalomyelitis in transgenic mice expressing ovalbumin in oligodendrocytes. Eur. J. Immunol. (2005). 80. Dezfouli,S., Hatzinisiriou,I., & Ralph,S.J. Use of cytokines in cancer vaccines/immunotherapy: recent developments improve survival rates for patients with metastatic malignancy. Curr. Pharm. Des 11, 3511-3530 (2005). 81. Sasaki,S., Takeshita,F., Xin,K.Q., Ishii,N., & Okuda,K. Adjuvant formulations and delivery systems for DNA vaccines. Methods 31, 243-254 (2003). 82. Cui,Z., Han,S.J., Vangasseri,D.P., & Huang,L. Immunostimulation mechanism of LPD nanoparticle as a vaccine carrier. Mol. Pharm. 2, 22-28 (2005). 83. Godbey,W.T., Wu,K.K., & Mikos,A.G. Poly(ethylenimine) and its role in gene delivery. J. Control Release 60, 149-160 (1999). 84. Kircheis,R., Wightman,L., & Wagner,E. Design and gene delivery activity of modified polyethylenimines. Adv. Drug Deliv. Rev. 53, 341-358 (2001). 85. Jendrisak,J.J. & Burgess,R.R. A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II. Biochemistry 14, 4639-4645 (1975). 86. Bochner,B.R. & Ames,B.N. Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. J. Biol. Chem. 257, 9759-9769 (1982). 87. Boussif,O., Lezoualc'h,F., Zanta,M.A., Mergny,M.D., Scherman,D., Demeneix,B., & Behr,J.P. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U. S. A 92, 7297-7301 (1995). 88. Boussif,O., Zanta,M.A., & Behr,J.P. Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther. 3, 1074-1080 (1996). 89. Wightman,L., Kircheis,R., Rossler,V., Carotta,S., Ruzicka,R., Kursa,M., & Wagner,E. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J. Gene Med. 3, 362-372 (2001). 90. Brunner,S., Sauer,T., Carotta,S., Cotten,M., Saltik,M., & Wagner,E. Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther. 7, 401-407 (2000). 91. Brunner,S., Furtbauer,E., Sauer,T., Kursa,M., & Wagner,E. Overcoming the nuclear barrier: cell cycle independent nonviral gene transfer with linear polyethylenimine or electroporation. Mol. Ther. 5, 80-86 (2002). 92. Demeneix,B., Behr,J., Boussif,O., Zanta,M.A., Abdallah,B., & Remy,J. Gene transfer with lipospermines and polyethylenimines. Adv. Drug Deliv. Rev. 30, 85-95 (1998). 93. Godbey,W.T., Wu,K.K., & Mikos,A.G. Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc. Natl. Acad. Sci. U. S. A 96, 5177-5181 (1999). 94. Ogris,M., Steinlein,P., Kursa,M., Mechtler,K., Kircheis,R., & Wagner,E. The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells. Gene Ther. 5, 1425-1433 (1998). 95. Goula,D., Remy,J.S., Erbacher,P., Wasowicz,M., Levi,G., Abdallah,B., & Demeneix,B.A. Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system. Gene Ther. 5, 712-717 (1998). 96. Demeneix,B., Behr,J., Boussif,O., Zanta,M.A., Abdallah,B., & Remy,J. Gene transfer with lipospermines and polyethylenimines. Adv. Drug Deliv. Rev. 30, 85-95 (1998). 97. Pollard,H., Remy,J.S., Loussouarn,G., Demolombe,S., Behr,J.P., & Escande,D. Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. J. Biol. Chem. 273, 7507-7511 (1998). 98. Oh,Y.K., Suh,D., Kim,J.M., Choi,H.G., Shin,K., & Ko,J.J. Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA. Gene Ther. 9, 1627-1632 (2002). 99. Kichler,A., Leborgne,C., Coeytaux,E., & Danos,O. Polyethylenimine-mediated gene delivery: a mechanistic study. J. Gene Med. 3, 135-144 (2001). 100. Densmore,C.L., Orson,F.M., Xu,B., Kinsey,B.M., Waldrep,J.C., Hua,P., Bhogal,B., & Knight,V. Aerosol delivery of robust polyethyleneimine-DNA complexes for gene therapy and genetic immunization. Mol. Ther. 1, 180-188 (2000). 101. Brunner,S., Furtbauer,E., Sauer,T., Kursa,M., & Wagner,E. Overcoming the nuclear barrier: cell cycle independent nonviral gene transfer with linear polyethylenimine or electroporation. Mol. Ther. 5, 80-86 (2002). 102. Brunner,S., Furtbauer,E., Sauer,T., Kursa,M., & Wagner,E. Overcoming the nuclear barrier: cell cycle independent nonviral gene transfer with linear polyethylenimine or electroporation. Mol. Ther. 5, 80-86 (2002). 103. Brunner,S., Sauer,T., Carotta,S., Cotten,M., Saltik,M., & Wagner,E. Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther. 7, 401-407 (2000). 104. Brunner,S., Furtbauer,E., Sauer,T., Kursa,M., & Wagner,E. Overcoming the nuclear barrier: cell cycle independent nonviral gene transfer with linear polyethylenimine or electroporation. Mol. Ther. 5, 80-86 (2002). 105. Kircheis,R., Ostermann,E., Wolschek,M.F., Lichtenberger,C., Magin-Lachmann,C., Wightman,L., Kursa,M., & Wagner,E. Tumor-targeted gene delivery of tumor necrosis factor-alpha induces tumor necrosis and tumor regression without systemic toxicity. Cancer Gene Ther. 9, 673-680 (2002). 106. Kircheis,R., Wightman,L., Kursa,M., Ostermann,E., & Wagner,E. Tumor-targeted gene delivery: an attractive strategy to use highly active effector molecules in cancer treatment. Gene Ther. 9, 731-735 (2002). 107. Kircheis,R., Blessing,T., Brunner,S., Wightman,L., & Wagner,E. Tumor targeting with surface-shielded ligand--polycation DNA complexes. J. Control Release 72, 165-170 (2001). 108. Kircheis,R., Schuller,S., Brunner,S., Ogris,M., Heider,K.H., Zauner,W., & Wagner,E. Polycation-based DNA complexes for tumor-targeted gene delivery in vivo. J. Gene Med. 1, 111-120 (1999). 109. Kircheis,R., Kichler,A., Wallner,G., Kursa,M., Ogris,M., Felzmann,T., Buchberger,M., & Wagner,E. Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Ther. 4, 409-418 (1997). 110. Kircheis,R., Kichler,A., Wallner,G., Kursa,M., Ogris,M., Felzmann,T., Buchberger,M., & Wagner,E. Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Ther. 4, 409-418 (1997). 111. Hsu,P.Y. & Yang,Y.W. Effect of polyethylenimine on recombinant adeno-associated virus mediated insulin gene therapy. J. Gene Med. 7, 1311-1321 (2005). 112. Regnstrom,K., Ragnarsson,E.G., Koping-Hoggard,M., Torstensson,E., Nyblom,H., & Artursson,P. PEI - a potent, but not harmless, mucosal immuno-stimulator of mixed T-helper cell response and FasL-mediated cell death in mice. Gene Ther. 10, 1575-1583 (2003). 113. McDonnell,W.M. & Askari,F.K. DNA vaccines. N. Engl. J. Med. 334, 42-45 (1996). 114. Fynan,E.F., Webster,R.G., Fuller,D.H., Haynes,J.R., Santoro,J.C., & Robinson,H.L. DNA vaccines: a novel approach to immunization. Int. J. Immunopharmacol. 17, 79-83 (1995). 115. Ulmer,J.B., DeWitt,C.M., Chastain,M., Friedman,A., Donnelly,J.J., McClements,W.L., Caulfield,M.J., Bohannon,K.E., Volkin,D.B., & Evans,R.K. Enhancement of DNA vaccine potency using conventional aluminum adjuvants. Vaccine 18, 18-28 (1999). 116. Kim,J.J., Nottingham,L.K., Wilson,D.M., Bagarazzi,M.L., Tsai,A., Morrison,L.D., Javadian,A., Chalian,A.A., Agadjanyan,M.G., & Weiner,D.B. Engineering DNA vaccines via co-delivery of co-stimulatory molecule genes. Vaccine 16, 1828-1835 (1998). 117. Marwick,C. Exciting potential of DNA vaccines explored. JAMA 273, 1403-1404 (1995). 118. Liu,M.A. & Ulmer,J.B. Human clinical trials of plasmid DNA vaccines. Adv. Genet. 55, 25-40 (2005). 119. Baylor,N.W., Egan,W., & Richman,P. Aluminum salts in vaccines--US perspective. Vaccine 20 Suppl 3, S18-S23 (2002). 120. De Smedt,T., Pajak,B., Muraille,E., Lespagnard,L., Heinen,E., De Baetselier,P., Urbain,J., Leo,O., & Moser,M. Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo. J. Exp. Med. 184, 1413-1424 (1996). 121. Bowie,A. & O'Neill,L.A. Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries. Biochem. Pharmacol. 59, 13-23 (2000). 122. Allison,T.J. & Garboczi,D.N. Structure of gammadelta T cell receptors and their recognition of non-peptide antigens. Mol. Immunol. 38, 1051-1061 (2002). 123. Lien,E. & Golenbock,D.T. Adjuvants and their signaling pathways: beyond TLRs. Nat. Immunol. 4, 1162-1164 (2003). 124. Goldszmid,R.S., Idoyaga,J., Bravo,A.I., Steinman,R., Mordoh,J., & Wainstok,R. Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma. J. Immunol. 171, 5940-5947 (2003). 125. Hernandez,J., Aung,S., Redmond,W.L., & Sherman,L.A. Phenotypic and functional analysis of CD8(+) T cells undergoing peripheral deletion in response to cross-presentation of self-antigen. J. Exp. Med. 194, 707-717 (2001). 126. Nowak,A.K., Lake,R.A., Marzo,A.L., Scott,B., Heath,W.R., Collins,E.J., Frelinger,J.A., & Robinson,B.W. Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation, cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells. J. Immunol. 170, 4905-4913 (2003). 127. Bonifaz,L.C., Bonnyay,D.P., Charalambous,A., Darguste,D.I., Fujii,S., Soares,H., Brimnes,M.K., Moltedo,B., Moran,T.M., & Steinman,R.M. In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination. J. Exp. Med. 199, 815-824 (2004). 128. Shirakawa,T., Hamada,K., Zhang,Z., Okada,H., Tagawa,M., Kamidono,S., Kawabata,M., & Gotoh,A. A cox-2 promoter-based replication-selective adenoviral vector to target the cox-2-expressing human bladder cancer cells. Clin. Cancer Res. 10, 4342-4348 (2004). 129. Lu,B., Makhija,S.K., Nettelbeck,D.M., Rivera,A.A., Wang,M., Komarova,S., Zhou,F., Yamamoto,M., Haisma,H.J., Alvarez,R.D., Curiel,D.T., & Zhu,Z.B. Evaluation of tumor-specific promoter activities in melanoma. Gene Ther. 12, 330-338 (2005). 130. Zhu,Z.B., Makhija,S.K., Lu,B., Wang,M., Kaliberova,L., Liu,B., Rivera,A.A., Nettelbeck,D.M., Mahasreshti,P.J., Leath,C.A., III, Yamaoto,M., Alvarez,R.D., & Curiel,D.T. Transcriptional targeting of adenoviral vector through the CXCR4 tumor-specific promoter. Gene Ther. 11, 645-648 (2004). 131. Zhu,Z.B., Makhija,S.K., Lu,B., Wang,M., Kaliberova,L., Liu,B., Rivera,A.A., Nettelbeck,D.M., Mahasreshti,P.J., Leath,C.A., Barker,S., Yamaoto,M., Li,F., Alvarez,R.D., & Curiel,D.T. Transcriptional targeting of tumors with a novel tumor-specific survivin promoter. Cancer Gene Ther. 11, 256-262 (2004). 132. Nettelbeck,D.M., Rivera,A.A., Davydova,J., Dieckmann,D., Yamamoto,M., & Curiel,D.T. Cyclooxygenase-2 promoter for tumour-specific targeting of adenoviral vectors to melanoma. Melanoma Res. 13, 287-292 (2003). 133. Yamamoto,M., Davydova,J., Wang,M., Siegal,G.P., Krasnykh,V., Vickers,S.M., & Curiel,D.T. Infectivity enhanced, cyclooxygenase-2 promoter-based conditionally replicative adenovirus for pancreatic cancer. Gastroenterology 125, 1203-1218 (2003). 134. Godbey,W.T. & Atala,A. Directed apoptosis in Cox-2-overexpressing cancer cells through expression-targeted gene delivery. Gene Ther. 10, 1519-1527 (2003). 135. Greenberger,S., Shaish,A., Varda-Bloom,N., Levanon,K., Breitbart,E., Goldberg,I., Barshack,I., Hodish,I., Yaacov,N., Bangio,L., Goncharov,T., Wallach,D., & Harats,D. Transcription-controlled gene therapy against tumor angiogenesis. J. Clin. Invest 113, 1017-1024 (2004). 136. Moore,M.W., Carbone,F.R., & Bevan,M.J. Introduction of soluble protein into the class I pathway of antigen processing and presentation. Cell 54, 777-785 (1988). 137. Karttunen,J., Sanderson,S., & Shastri,N. Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc. Natl. Acad. Sci. U. S. A 89, 6020-6024 (1992). 138. Sanderson,S. & Shastri,N. LacZ inducible, antigen/MHC-specific T cell hybrids. Int. Immunol. 6, 369-376 (1994). 139. Thery,C. & Amigorena,S. The cell biology of antigen presentation in dendritic cells. Curr. Opin. Immunol. 13, 45-51 (2001). 140. Hockenbery,D.M., Oltvai,Z.N., Yin,X.M., Milliman,C.L., & Korsmeyer,S.J. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75, 241-251 (1993). 141. Perticarari,S., Presani,G., & Banfi,E. A new flow cytometric assay for the evaluation of phagocytosis and the oxidative burst in whole blood. J. Immunol. Methods 170, 117-124 (1994). 142. KAHLER,R.L. & GUZE,L.B. Evaluation of the Griess nitrite test as a method for the recognition of urinary tract infection. J. Lab Clin. Med. 49, 934-937 (1957). 143. Matsue,H., Edelbaum,D., Shalhevet,D., Mizumoto,N., Yang,C., Mummert,M.E., Oeda,J., Masayasu,H., & Takashima,A. Generation and function of reactive oxygen species in dendritic cells during antigen presentation. J. Immunol. 171, 3010-3018 (2003). 144. Zhao,R., Masayasu,H., & Holmgren,A. Ebselen: a substrate for human thioredoxin reductase strongly stimulating its hydroperoxide reductase activity and a superfast thioredoxin oxidant. Proc. Natl. Acad. Sci. U. S. A 99, 8579-8584 (2002). 145. Zhao,R. & Holmgren,A. A novel antioxidant mechanism of ebselen involving ebselen diselenide, a substrate of mammalian thioredoxin and thioredoxin reductase. J. Biol. Chem. 277, 39456-39462 (2002). 146. Schewe,C., Schewe,T., & Wendel,A. Strong inhibition of mammalian lipoxygenases by the antiinflammatory seleno-organic compound ebselen in the absence of glutathione. Biochem. Pharmacol. 48, 65-74 (1994). 147. Hattori,R., Inoue,R., Sase,K., Eizawa,H., Kosuga,K., Aoyama,T., Masayasu,H., Kawai,C., Sasayama,S., & Yui,Y. Preferential inhibition of inducible nitric oxide synthase by ebselen. Eur. J. Pharmacol. 267, R1-R2 (1994). 148. Ulmer,J.B., DeWitt,C.M., Chastain,M., Friedman,A., Donnelly,J.J., McClements,W.L., Caulfield,M.J., Bohannon,K.E., Volkin,D.B., & Evans,R.K. Enhancement of DNA vaccine potency using conventional aluminum adjuvants. Vaccine 18, 18-28 (1999). 149. Wang,S., Liu,X., Fisher,K., Smith,J.G., Chen,F., Tobery,T.W., Ulmer,J.B., Evans,R.K., & Caulfield,M.J. Enhanced type I immune response to a hepatitis B DNA vaccine by formulation with calcium- or aluminum phosphate. Vaccine 18, 1227-1235 (2000). 150. HogenEsch,H. Mechanisms of stimulation of the immune response by aluminum adjuvants. Vaccine 20 Suppl 3, S34-S39 (2002). 151. Baylor,N.W., Egan,W., & Richman,P. Aluminum salts in vaccines--US perspective. Vaccine 20 Suppl 3, S18-S23 (2002). 152. Rosado-Vallado,M., Mut-Martin,M., Garcia-Miss,M.R., & Dumonteil,E. Aluminium phosphate potentiates the efficacy of DNA vaccines against Leishmaniamexicana. Vaccine 23, 5372-5379 (2005). 153. Goldszmid,R.S., Idoyaga,J., Bravo,A.I., Steinman,R., Mordoh,J., & Wainstok,R. Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma. J. Immunol. 171, 5940-5947 (2003). 154. Hernandez,J., Aung,S., Redmond,W.L., & Sherman,L.A. Phenotypic and functional analysis of CD8(+) T cells undergoing peripheral deletion in response to cross-presentation of self-antigen. J. Exp. Med. 194, 707-717 (2001). 155. Rajananthanan,P., Attard,G.S., Sheikh,N.A., & Morrow,W.J. Evaluation of novel aggregate structures as adjuvants: composition, toxicity studies and humoral responses. Vaccine 17, 715-730 (1999). 156. Rajananthanan,P., Attard,G.S., Sheikh,N.A., & Morrow,W.J. Novel aggregate structure adjuvants modulate lymphocyte proliferation and Th1 and Th2 cytokine profiles in ovalbumin immunized mice. Vaccine 18, 140-152 (1999). 157. Hsu,P.Y. & Yang,Y.W. Effect of polyethylenimine on recombinant adeno-associated virus mediated insulin gene therapy. J. Gene Med. 7, 1311-1321 (2005). 158. Bowie,A. & O'Neill,L.A. Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries. Biochem. Pharmacol. 59, 13-23 (2000). 159. Lambeth,J.D. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4, 181-189 (2004). 160. Hsu,P.Y. & Yang,Y.W. Effect of polyethylenimine on recombinant adeno-associated virus mediated insulin gene therapy. J. Gene Med. 7, 1311-1321 (2005). 161. Hockenbery,D.M., Oltvai,Z.N., Yin,X.M., Milliman,C.L., & Korsmeyer,S.J. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75, 241-251 (1993). 162. Perticarari,S., Presani,G., & Banfi,E. A new flow cytometric assay for the evaluation of phagocytosis and the oxidative burst in whole blood. J. Immunol. Methods 170, 117-124 (1994). 163. KAHLER,R.L. & GUZE,L.B. Evaluation of the Griess nitrite test as a method for the recognition of urinary tract infection. J. Lab Clin. Med. 49, 934-937 (1957). 164. Karttunen,J., Sanderson,S., & Shastri,N. Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc. Natl. Acad. Sci. U. S. A 89, 6020-6024 (1992). 165. Sanderson,S. & Shastri,N. LacZ inducible, antigen/MHC-specific T cell hybrids. Int. Immunol. 6, 369-376 (1994). 166. Negulescu,P.A., Shastri,N., & Cahalan,M.D. Intracellular calcium dependence of gene expression in single T lymphocytes. Proc. Natl. Acad. Sci. U. S. A 91, 2873-2877 (1994). 167. Matsue,H., Edelbaum,D., Shalhevet,D., Mizumoto,N., Yang,C., Mummert,M.E., Oeda,J., Masayasu,H., & Takashima,A. Generation and function of reactive oxygen species in dendritic cells during antigen presentation. J. Immunol. 171, 3010-3018 (2003). 168. Zhao,R., Masayasu,H., & Holmgren,A. Ebselen: a substrate for human thioredoxin reductase strongly stimulating its hydroperoxide reductase activity and a superfast thioredoxin oxidant. Proc. Natl. Acad. Sci. U. S. A 99, 8579-8584 (2002). 169. Zhao,R. & Holmgren,A. A novel antioxidant mechanism of ebselen involving ebselen diselenide, a substrate of mammalian thioredoxin and thioredoxin reductase. J. Biol. Chem. 277, 39456-39462 (2002). 170. Schewe,C., Schewe,T., & Wendel,A. Strong inhibition of mammalian lipoxygenases by the antiinflammatory seleno-organic compound ebselen in the absence of glutathione. Biochem. Pharmacol. 48, 65-74 (1994). 171. Hattori,R., Inoue,R., Sase,K., Eizawa,H., Kosuga,K., Aoyama,T., Masayasu,H., Kawai,C., Sasayama,S., & Yui,Y. Preferential inhibition of inducible nitric oxide synthase by ebselen. Eur. J. Pharmacol. 267, R1-R2 (1994). 172. Bowie,A. & O'Neill,L.A. Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries. Biochem. Pharmacol. 59, 13-23 (2000). 173. Lien,E. & Golenbock,D.T. Adjuvants and their signaling pathways: beyond TLRs. Nat. Immunol. 4, 1162-1164 (2003). 174. Russell,J.P., Engiles,J.B., & Rothstein,J.L. Proinflammatory mediators and genetic background in oncogene mediated tumor progression. J. Immunol. 172, 4059-4067 (2004). 175. Colle,J.H., Falanga,P.B., Singer,M., Hevin,B., & Milon,G. Quantitation of messenger RNA by competitive RT-PCR: a simplified read out assay. J. Immunol. Methods 210, 175-184 (1997). 176. Ulmer,J.B., D | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34853 | - |
dc.description.abstract | DNA疫苗雖然比傳統活菌減毒疫苗或死菌疫苗更為安全且穩定,但由於單獨接種DNA疫苗效力有限,因此必須在疫苗佐劑的輔助下才能引起長期有效的免疫反應。在本論文中,我們嘗試使用大家熟知具有良好基因遞送能力的陽離子聚合物-聚乙二烯胺 (polyethylenimine;PEI) 作為DNA疫苗佐劑並於試管中與活體中評估其發展潛能。
首先,我們以MTT (3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) 測定法得知樹突狀細胞對於PEI的毒性耐受度為8 μg/ml,並使用界面電位測定儀與粒徑分析儀分析PEI/DNA複合物在不同N/P比例之物化特性,發現PEI/DNA複合物在N/P=10時,不僅劑型最穩定,對樹突狀細胞的基因轉染效率也最好。我們亦使用流式細胞儀分析樹突狀細胞經8 μg/ml PEI刺激24小時後的成熟活化情形,結果顯示樹突狀細胞不僅大量表現CD40、CD80、CD86等共刺激分子,其抗原捕捉能力也顯著下降,符合樹突狀細胞成熟活化的表型。細胞螢光免疫染色法和西方墨點法的結果顯示轉錄因子NFκB會在PEI的刺激下活化且轉位到細胞核,可能就是造成樹突狀細胞活化的主因。同時,我們使用2’,7’-dichlorofluorescein diacetate (DCFH-DA) 和hydroethidine (HE) 等螢光探針經流式細胞儀分析,確認了PEI刺激樹突狀細胞產生H2O2和O2-等活性氧系 (reactive oxygen species;ROS) 的情形。使用抗氧化分子ebselen會抑制ROS在樹突狀細胞的生成並降低樹突狀細胞的抗原呈現能力,再次證明了ROS在活化樹突狀細胞中扮演了重要角色。 我們使用PEI將pAc-neo-OVA質體DNA遞送至樹突狀細胞內後,發現樹突狀細胞不僅可以經由MHC class I分子呈現OVA抗原,還可引發抗原專一性的第一型免疫反應,有效的活化CD8+且具OVA專一性T細胞受器 (OVA-specific TCR) 的B3Z細胞。有別於其他基因遞送載體,我們亦從抗氧化分子ebselen的實驗證明了PEI在遞送基因的同時,能夠活化樹突狀細胞的獨特性質是其能有效引起免疫反應的原因之一。接著,我們以反轉錄聚合酵素鏈鎖反應 (RT-PCR) 分析經PEI刺激後樹突狀細胞mRNA表現量的消長情形,結果顯示僅第二型環氧化酶 (COX-2) 有大量表現的情形,卻未能觀察到其他發炎前期細胞激素如IL-1β、IL-6、 IL-12和 TNF-α等基因表現的增加。這些結果顯示PEI引起樹突狀細胞成熟活化或許並非經由模式辨識受器 (pattern recognition receptors;PRRs) 所媒介,而是經由ROS直接引起發炎反應。 在動物實驗中,我們於小鼠腳掌經皮下/皮間的途徑接種疫苗,發現接種PEI/DNA複合物疫苗的小鼠,其接種位置的樹突狀細胞會呈現OVA抗原並移行到近端淋巴結。為了證實這些樹突狀細胞能有效活化T細胞,我們也以B3Z細胞進行小鼠體內OVA專一性免疫反應鑑定 (in vivo B3Z assay)。實驗結果顯示PEI/DNA複合物疫苗能有效活化第一型免疫反應,而這些活化的樹突狀細胞也只會滯留在近端淋巴結,並不會進一步移行到脾臟。同時我們亦使用terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL) assay確認了PEI在疫苗接種位置造成的組織傷害低於FDA核准可使用在人體上的疫苗佐劑-磷酸鋁 (aluminum phosphate;AlPi) ,彰顯其安全性。接著我們使用酵素連結免疫吸附法 (enzyme linked immunosorbant assay;ELISA) 分析小鼠接種疫苗後體內抗OVA抗體的生成情形與抗體亞型轉換的情形以確認免疫系統偏極化 (immunity polarization) 的現象。而活體內的細胞毒殺試驗 (in vivo CTL assay) 亦證實PEI/DNA複合物疫苗能在小鼠體內有效活化OVA專一性毒殺型T細胞,建立長期的OVA專一性細胞免疫反應。此外,無論我們將PEI/DNA複合物疫苗應用作為預防性或治療性的癌症疫苗,都可得到不錯的療效,達到有效延緩EG7-OVA腫瘤在小鼠體內的生長速度以及提高小鼠罹癌後的存活率之目的。 為了將質體DNA作進一步的改造以期能強化DNA疫苗的腫瘤專一性靶向能力,減少治療基因在正常細胞表現的機會以降低其副作用,我們以惡性黑色素細胞瘤B16F10小鼠腫瘤模式篩選出最具療效的共刺激分子基因CD86和自殺基因caspase 3後,再選用多數腫瘤細胞會大量表現的第二型環氧化酶之驅動子 (COX-2 promoter) 驅動上述兩個治療基因並構築在重組腺相關病毒 (rAAV) 的inverted terminal repeat (ITR) 之間,希望能在轉錄階層靶向表現在B16F10腫瘤細胞上。實驗結果顯示在PEI的輔助下,此DNA疫苗的確能有效抑制惡性黑色素細胞瘤B16F10在小鼠體內的生長。 總而言之,本研究不僅提供了PEI刺激樹突狀細胞成熟活化的作用機轉、在活體實驗中發現PEI可在低毒性範圍達到有效轉染治療基因並成功建立第一型與第二型抗原專一性免疫反應,也在小鼠腫瘤模式中證實了應用PEI作為預防性或治療性DNA癌症疫苗佐劑的可行性。 | zh_TW |
dc.description.abstract | DNA-based vaccines are safer and more stable than conventional protein vaccines containing live-attenuated or killed bacteria/viruses. However, the immunogenicity of this new generation vaccine is often poor when administered alone, and it often requires incorporation with the adjuvants to enhance the immune response. In the present study, polyethylenimine (PEI), a cationic polymer commonly used in gene delivery, was examined in vitro and in vivo for the potential of being a candidate adjuvant for DNA vaccines.
The 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the direct cytotoxicity of PEI in the bone marrow-derived dendritic cells (BMDCs), the most potent antigen presenting cells (APCs), from C57BL/6J mice. The physicochemical properties, including size and zeta potential, and the transfection efficiency of the PEI-DNA complexes were measured at various N/P ratios. Maturation of dendritic cells is a crucial step in the initiation of an adaptive immune response. To examine the direct immuno-stimulatory effect of PEI on the APCs, the BMDCs at Day 6 were treated with PEI (8 μg/ml) for 24 hrs, followed by flow cytometric analysis of co-stimulatory molecules expression. Treatment of BMDCs with PEI for 24 hrs increased the expression of maturation markers (CD40, CD80, and CD86) of CD11c+ dendritic cells and decreased their antigen-capturing capability. Fluorescence microscopic examination and Western blot analysis demonstrated that treatment of BMDCs with PEI resuled in translocation of transcription factor NFκB (Nuclear Factor kappa B) to the nucleus. These results illustrated the involvement of NFκB signaling in immunogenicity elicited by PEI. The possible roles of oxidative stress on immunogenicity after treatment of PEI were examined in BMDCs by measuring the production of reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and superoxide (O2‾), using 2’,7’-dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE). Treatment of BMDCs with PEI exhibited a time- and dose-dependent increase in the fluorescent products 2’,7’-dichlorofluorescein (DCF) and ethidium bromide (EB), peaked at 32 μg/ml PEI. Addition of ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one), a substrate for thioredoxin reductase, suppressed the increased ROS production induced by PEI with concomitant decreased expression of co-stimulatory molecules in the BMDCs. Transfection of BMDCs with pAc-neo-OVA plasmid DNA-PEI complexes resulted in MHC-I restricted OVA presentation, as assayed by B3Z cells, a CD8+ murine T cell hybridoma expresses OVA-specific T cell receptors and transfected with LacZ reporter gene under the control of NFAT (Nuclear Factor of Activated T cells) promoter. Pretreatment of BMDCs with ebselen decreased B3Z activation without changes in MHC-I restricted antigen presentation, suggesting the roles of the ROS in adjuvant-mediated DC maturation. Analysis of the pro-inflammatory effect of PEI by reverse transcription-polymerase chain reaction (RT-PCR) illustrated that treatment of BMDCs with PEI induced significant up-regulation of COX-2 mRNA but not other pro-inflammatory cytokines tested, including IL-1β, IL-6, IL-12, and TNF-α, etc.. These results suggested that signaling mediated by toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs) may not be essential for PEI-mediated adjuvanticity. The in vivo adjuvant effect of PEI for DNA vaccines was examined in EL4/EG7-OVA syngeneic mouse lymphoma model. Immunization was carried out in C57BL/6J mice by subcutaneous/intradermal inoculation of pAc-neo-OVA plasmid DNA-PEI complexes into the footpads of the animals, followed by periodic measurements of tumor sizes, enzyme linked immunosorbant assay (ELISA) for the antibody response, and determination of immunity polarization. Our results showed that DNA vaccination when incorporated with PEI significantly reduced tumor growth, increased the survival rate of the animals, and elevated the anti-OVA antibody titers. Flow cytometric analysis of lymphoid organs after vaccination showed increased numbers of OVA-specific CD11c+ DCs migrating to draining lymph nodes in those animals injected with DNA-PEI complexes. The terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL) assay of tissue sections examined at vaccination sites also demonstrated the safety in using PEI as an adjuvant with lower toxicity compared to FDA approved adjuvant-aluminum phosphate (AlPi). The in vivo B3Z assay demonstrated that immunization of mice with DNA-PEI complexes resulted in MHC class I-restricted antigen presentation predominantly in the draining lymph nodes. The in vivo adjuvant potency of PEI was further confirmed by the significant cytotoxic effect of OVA-specific cytotoxic T lymphocytes (CTLs) in the animals immunized with pAc-neo-OVA DNA-PEI complexes, exhibiting higher protection from EG7-OVA tumor challenge. To further examine the in vivo adjuvant effect of PEI for immuno-modulating DNA vaccines, studies were extended to examine the therapeutic effect of DNA vaccination in B16F10 melanoma-bearing animals. Significant anti-tumor effect was observed after immunization of tumor-bearing mice with the DNA-PEI complexes containing the mIL-2, h caspase3, mCD80, or mCD86 genes. In an attempt to transcriptionally target tumors overexpressing COX-2, plasmids were constructed by inserting the mCD86 and C2P-caspase3△9 genes, under the control of COX-2 promoter, into the recombinant adeno-associated virus (rAAV) vectors, followed by immunization in the melanoma-bearing mice. Complexation of these constructs with PEI was shown to significantly inhibit tumor growth in the B16F10 melanoma-bearing mice. In conclusion, strategies were developed in the present study employing PEI as an adjuvant for generation of effective DNA vaccination. Our results demonstrated that PEI activated BMDCs, triggered NFκB signaling cascade via ROS production, stimulated antigen-specific type I and type II immune responses, leading to effective tumor protection in tumor-bearing animals, both for prophylactic and therapeutic DNA vaccines. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T06:35:38Z (GMT). No. of bitstreams: 1 ntu-95-R92423012-1.pdf: 1933255 bytes, checksum: 18cde2f7fde2cab3053ae0eb9da7ce4e (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 壹、文獻回顧 1
一、免疫系統(The immune system) 1 二、DNA疫苗(DNA vaccines) 3 三、疫苗佐劑(Vaccine adjuvants) 6 四、聚乙二烯胺 ( Polyethylenimine ) 7 貳、研究動機與實驗設計 9 參、材料與方法 12 材料 12 方法 17 A-1 細胞培養 17 A-2 質體DNA (plasmid DNA) 的製備 18 A-3 Total RNA之抽取、定量與分析 20 A-4 蛋白質的萃取、定量與分析 22 A-5 動物實驗 23 一、PEI與PEI/DNA複合物之特性分析 25 1-1 樹突狀細胞的抽取與誘導 25 1-2 PEI之細胞毒性測定 26 1-3 細胞表面電荷 (zeta potential) 之測定 26 1-4 PEI/DNA複合物粒徑大小與表面電荷之測定 27 1-5 不同比例的PEI/DNA複合物之基因轉染 ( transfection ) 效率分析 27 二、PEI引起樹突狀細胞活化的免疫機制探討 28 2-1 樹突狀細胞成熟活化之分析 28 2-2 NFκB之活化程度分析 29 2-3 PEI所引起的活性氧系 ( Reactive Oxygen Species;ROS) 分析 29 2-4 樹突狀細胞的抗原呈現強度分析 ( B3Z assay ) 30 2-5 活性氧系的抑制方法 31 三、治療性疫苗在小鼠EG7-OVA腫瘤模式之應用 31 3-1 DNA疫苗的配方、施打方式與施打時程 (immunization schedule) 31 四、預防性疫苗在小鼠EL4 / EG7-OVA腫瘤模式之應用 32 4-1 DNA疫苗的配方、施打方式與施打時程 32 4-2 接種疫苗所引起的急性免疫反應之評估 33 4-3 接種疫苗所引起的長期免疫反應之評估 35 五、治療性疫苗在小鼠B16F10腫瘤模式之應用 37 5-1 DNA疫苗的配方、施打方式與施打時程 37 5-2 質體DNA ( plasmid DNA) 之構築 39 數據分析 41 肆、結果 42 第一部分:PEI應用為DNA疫苗佐劑之作用機轉探討 42 一、PEI在高濃度具有細胞毒性,在低濃度卻可促進樹突狀細胞生長分裂。 42 二、PEI會對樹突狀細胞的表面電位造成影響 43 三、PEI/DNA複合物在表面電荷為電中性時會形成較大的顆粒 43 四、PEI/DNA複合物在樹突狀細胞的基因轉染效率 44 五、PEI可在無毒性範圍引起樹突狀細胞的成熟活化 44 六、PEI會促使NFκB活化並轉位到細胞核 46 七、PEI會刺激樹突狀細胞產生ROS,造成細胞壓力 46 八、PEI/DNA複合物可使樹突狀細胞經第一型MHC分子呈現OVA抗原 47 九、Ebselen會降低PEI在樹突狀細胞產生ROS的情形,並抑制樹突狀細胞的活化 48 十、Ebselen不改變PEI/DNA複合物在樹突狀細胞的基因轉染率,卻使得樹突狀細胞呈現抗原的能力下降 49 十一、PEI會刺激樹突狀細胞產生發炎反應 50 第二部分:治療性疫苗在小鼠EG7-OVA腫瘤模式之應用 51 一、以PEI作為DNA疫苗佐劑可延緩EG7-OVA腫瘤在小鼠的生長速度及提高小鼠的存活率 51 二、接種不同劑型的DNA疫苗後,EG7-OVA腫瘤微環境細胞激素基因的表現情形 52 第三部分:預防性疫苗在小鼠EL4 / EG7-OVA腫瘤模式之應用 53 一、PEI/DNA複合物能將OVA基因轉染到疫苗注射位置的樹突狀細胞上,並使之活化而移行到近端淋巴結 53 二、以PEI作為DNA疫苗佐劑可使移行到近端淋巴結的樹突狀細胞活化B3Z細胞 55 三、接種PEI/DNA複合物疫苗之小鼠體內會顯著的產生OVA專一性毒殺型T細胞 55 四、長期接種疫苗後,小鼠體內抗OVA抗體的生成情形及免疫系統的偏向 56 五、接種PEI/DNA複合物疫苗之小鼠可有效的排斥EG7-OVA腫瘤並延緩腫瘤的生長 57 第四部分:治療性疫苗在小鼠B16F10腫瘤模式之應用 58 伍、討論 61 一、關於PEI應用為DNA疫苗佐劑的作用機轉 61 二、關於PEI/DNA複合物疫苗於活體應用的情形 64 三、關於癌症基因治療的延伸實驗 67 陸、結論 69 柒、參考資料 70 捌、圖表 94 玖、附錄 126 | |
dc.language.iso | zh-TW | |
dc.title | 聚乙二烯胺作為DNA疫苗佐劑之研究 | zh_TW |
dc.title | Studies of polyethylenimine as a DNA vaccine adjuvant | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 江伯倫,伍安怡,陳小梨 | |
dc.subject.keyword | 聚乙二烯胺,DNA疫苗,疫苗佐劑,癌症免疫治療, | zh_TW |
dc.subject.keyword | polyethylenimine,DNA vaccine,adjuvant,cancer immunotherapy, | en |
dc.relation.page | 128 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-01-12 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥學研究所 | zh_TW |
顯示於系所單位: | 藥學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-95-1.pdf 目前未授權公開取用 | 1.89 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。