臺灣博碩士論文加值系統

口服免疫耐受性 (oral tolerance) 是口服抗原引發免疫力的一個阻礙。過去有些有效的黏膜佐劑如CT和LT曾被研究在免疫增強的功效。但由於它們本身帶有毒性，因此使用上是倍受考慮的。從我們實驗室過去的研究發現，由靈芝(Ganoderma lucidum) 所萃取出的多醣，經口服確實是具有抗癌和調節免疫系統的作用，雖然它在人體的吸收及代謝仍然不明，但這提供了靈芝多醣可以作用於腸道免疫系統的線索。因此，我們擬對靈芝多醣以口服的方式，扮演免疫調節劑或黏膜佐劑的角色作進一步的探討。
我們使用兩種抗原，一是以聚合物包裹的OVA顆粒，一是肺炎雙球莢膜多醣疫苗，分別以皮下注射或口服的方式來引發小鼠抗體免疫反應。另外，我們將靈芝多醣加入小鼠日常飲水中以作為免疫調節劑之用，或將多醣混合抗原同時給予小鼠服用以作為黏膜佐劑之用。
就靈芝多醣長期服用作為免疫調節劑的層面來看，靈芝多醣可增進以皮下注射OVA顆粒或肺炎雙球莢膜多醣所引發在血液中的抗體量；亦可促進以口服OVA顆粒或肺炎雙球莢膜多醣所引起肺部抗體IgA的表現量；但卻會抑制以口服上述的兩種抗原所引發在血液中抗體IgG的產生。奇妙的是，將靈芝多醣以免疫調節劑的方式作用在20週大的小鼠身上，它可以突破免疫耐受性的禁錮，讓直接口服ovalbumin蛋白的小鼠在血液中產生高量的抗體IgG。
若就靈芝多醣用作與抗原混合的黏膜佐劑的層面來看，靈芝多醣會抑制以口服OVA顆粒所引發在血液中的抗體IgG，但卻會促進以此方式所引發在腸道及肺部抗體IgA的分泌。另外，靈芝多醣會增進以口服肺炎雙球莢膜多醣所引發在血液中IgM的表現量，但對腸道及肺部中的抗體IgA則沒有影響。
此外，我們利用反轉錄聚合鏈反應 (RT-PCR) 來分析服用三天靈芝多醣的小鼠，其細胞激活素 (cytokines) 的表現情形。在所有分析的組織中，包括脾臟、腸道表皮層和固有層，在IL-1的表現皆呈促進的狀況，而其表現量的增加或許可解釋先前本實驗室的發現－口服靈芝多醣會活化自然殺手細胞的活性。另外，小鼠的脾臟及腸道的Peyer氏斑在靈芝多醣的作用下，前者的細胞激活素IFN- 和TGF-會隨多醣劑量增加而受抑制；但後者則相反成促進的情形。因此，由RT-PCR的結果可顯示出，靈芝多醣可能是透過細胞激活素的調控來發揮它的功效。

Oral tolerance is the obstacle for inducing immunity by oral route. There were some powerful mucosal adjuvants, such as CT and LT that had been studied for their capacity in immune potentiation. Due to their toxicity per se, their usage was carefully considered. In the previous study from our laboratory, it was shown that Ling-zhi polysaccharides isolated from Ganoderma lucidum had the effects on anti-tumor and modulating the immune system through oral administration, though their absorption by intestine and metabolism were unclear. This implied that Ling-zhi polysaccharides, in some way, might act on the intestinal immune system. We tried to identify the posibility of using Ling-zhi polysaccharides orally as an immunomodulator or a mucosal adjuvant.
We used two different antigens ovalbumin entrapped in PLGA (OVA particles) and Pneumovax 23 vaccine to induce humoral immune response in mice by subcutaneous immunization or by oral administration. Additionally, Ling-zhi polysaccharides were supplemented in daily drink for mice as an immunomodulator or mixed with antigens given orally as a mucosal adjuvant.
For an immunomodulator, Ling-zhi polysaccharides enhanced sera antibody production induced by subcutaneous immunization with OVA particles or Pneumovax 23 vaccine, and enhanced pulmonary IgA production induced by oral inoculation with OVA particles and Pneumovax 23 vaccine, but inhibited sera IgG production induced by oral inoculation with the same antigens. Marvelously, using Ling-zhi polysaccharides as an immunomodulator in 20-week-old mice, they broke oral tolerance and induced significant sera IgG production induced by oral administration with ovalbumin protein directly.
For a mucosal adjuvant, Ling-zhi polysaccharides inhibited sera IgG production but enhanced pulmonary and intestinal IgA secrection induced by oral administration with OVA particles. Additionally, it enhanced sera IgM production but had no influence on mucosal IgA secrection by oral inoculation with Pneumovax 23 vaccine.
In another way, we used reverse transcription and polymerase-chain-reaction (RT-PCR) to analyze the expression of some cytokines in mice fed with Ling-zhi polysaccharides for three days. In all the tissues we analyzed, including spleen、intestinal epithelium and lamina propria, IL-1beta was enhanced and its expression might be related to the induction of natural killer cell’s activity. Otherwise, the expressions of IFN-gamma and TGF-beta in spleen were inhibited with the increasing dose of Ling-zhi polysaccharides, while those in Peyer’s patch were opposite. Taken together, Ling-zhi polysaccharides did have their effects on intestinal immune system possibly through cytokine regulation.

中文摘要---------------------------------------------------- 1
英文摘要---------------------------------------------------- 2
縮寫表------------------------------------------------------ 4
圖表說明---------------------------------------------------- 5
緒論-------------------------------------------------------- 9
試藥及器材-------------------------------------------------- 13
方法-------------------------------------------------------- 17
實驗結果---------------------------------------------------- 35
討論-------------------------------------------------------- 41
參考資料---------------------------------------------------- 48

1) Moog F. The lining of the small intestine. Scientific American 1981, 245(5): 154-8.
2) Tomasi TB Jr. Mechanisms of immune regulation at mucosal surfaces. Reviews of Infectious Diseases 1983, 5 Suppl 4: S784-92.
3) McGhee JR. Mestecky J. Dertzbaugh MT. Eldridge JH. Hirasawa M. Kiyono H. The mucosal immune system: from fundamental concepts to vaccine development. Vaccine 1992, 10(2): 75-88.
4) Heel KA. McCauley RD. Papadimitriou JM. Hall JC. Review: Peyer''s patches. Journal of Gastroenterology & Hepatology 1997, 12(2): 122-36.
5) Strober W. Kelsall B. Marth T. Oral tolerance. Journal of Clinical Immunology 1998, 18(1): 1-30.
6) Ruedl C. Wolf H. Features of oral immunization. International Archives of Allergy & Immunology 1995, 108(4): 334-9.
7) Xin YY. Ming ZG. Peng GY. Jian A. Min LH. Safety of a live-attenuated Japanese encephalitis virus vaccine (SA14-14-2) for children. American Journal of Tropical Medicine & Hygiene 1988, 39(2): 214-7.
8) Kim SY. Doh HJ. Ahn JS. Ha YJ. Jang MH. Chung SI. Park HJ. Induction of mucosal and systemic immune response by oral immunization with H. pylori lysates encapsulated in poly(D,L-lactide-co-glycolide) microparticles. Vaccine 1999, 17(6): 607-16.
9) Moldoveanu Z. Novak M. Huang WQ. Gilley RM. Staas JK. Schafer D. Compans RW. Mestecky J. Oral immunization with influenza virus in biodegradable microspheres. Journal of Infectious Diseases 1993, 167(1): 84-90.
10) Takahashi I. Marinaro M. Kiyono H. Jackson RJ. Nakagawa I. Fujihashi K. Hamada S. Clements JD. Bost KL. McGhee JR. Mechanisms for mucosal immunogenicity and adjuvancy of Escherichia coli labile enterotoxin. Journal of Infectious Diseases 1996, 173(3): 627-35.
11) Marinaro M. Staats HF. Hiroi T. Jackson RJ. Coste M. Boyaka PN. Okahashi N. Yamamoto M. Kiyono H. Bluethmann H. et al. Mucosal adjuvant effect of cholera toxin in mice results from induction of T helper 2 (Th2) cells and IL-4. Journal of Immunology 1995, 155(10): 4621-9.
12) Robinson K. Chamberlain LM. Schofield KM. Wells JM. Le Page RW. Oral vaccination of mice against tetanus with recombinant Lactococcus lactis. Nature Biotechnology 1997, 15(7): 653-7.
13) Thanavala Y. Yang YF. Lyons P. Mason HS. Arntzen C. Immunogenicity of transgenic plant-derived hepatitis B surface antigen. Proceedings of the National Academy of Sciences of the United States of America 1995, 92(8): 3358-61.
14) Mason HS. Ball JM. Shi JJ. Jiang X. Estes MK. Arntzen CJ. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proceedings of the National Academy of Sciences of the United States of America 1996, 93(11): 5335-40.
15) Mason HS. Haq TA. Clements JD. Arntzen CJ. Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine 1998, 16(13): 1336-43.
16) . Ma SW. Zhao DL. Yin ZQ. Mukherjee R. Singh B. Qin HY. Stiller CR. Jevnikar AM. Transgenic plants expressing autoantigens fed to mice to induce oral immune tolerance. Nature Medicine 1997, 3(7): 793-6.
17) Bartnicki-Garcia S. Cell wall chemistry, morphogenesis, and taxonomy of fungi. Annual Review of Microbiology 1968, 22: 87-108.
18) . Jong SC. Birmingham JM. Medicinal benefits of the mushroom Ganoderma. Advances in Applied Microbiology 1992, 37: 101-34.
19) Lei LS. Lin ZB. Effect of Ganoderma polysaccharides on T cell subpopulations and production of interleukin 2 in mixed lymphocyte response. Yao Hsueh Hsueh Pao - Acta Pharmaceutica Sinica 1992. 27(5): 331-5.
20) Wang SY. Hsu ML. Hsu HC. Tzeng CH. Lee SS. Shiao MS. Ho CK. The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and T lymphocytes. International Journal of Cancer 1997, 70(6): 699-705.
21) Won SJ. Lin MT. Wu WL. Ganoderma tsugae mycelium enhances splenic natural killer cell activity and serum interferon production in mice. Japanese Journal of Pharmacology 1992. 59(2): 171-6.
22) Furusawa E., Chou S.C., Furusawa S., Hirazumi A., Dang Y. Antitumour activity of Ganoderma lucidum, an edible mushroom, on intraperitoneally implanted Lewis lung carcinoma in synergenic mice. Phytotherapy Research 1992, 6: 300-304.
23) 蔡勝發。(1999) 中藥靈芝抗老化及抗癌作用之研究。國立陽明大學生物藥學研究所碩士論文。
24) Sone Y., Okuda R., Wada N. Structures and antitumor activities of the polysaccharides isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum. Agric. Biol. Chem. 1985, 49(9): 2641-2653.
25) Desai MP. Labhasetwar V. Amidon GL. Levy RJ. Gastrointestinal uptake of biodegradable microparticles: effect of particle size. Pharmaceutical Research 1996. 13(12): 1838-45.
26) Jeffery H. Davis SS. O''Hagan DT. The preparation and characterization of poly(lactide-co-glycolide) microparticles. II. The entrapment of a model protein using a (water-in-oil)-in-water emulsion solvent evaporation technique. Pharmaceutical Research 1993, 10(3): 362-8.
27) Coombes AG. Yeh MK. Lavelle EC. Davis SS. The control of protein release from poly(DL-lactide co-glycolide) microparticles by variation of the external aqueous phase surfactant in the water-in oil-in water method. Journal of Controlled Release 1998, 52(3): 311-20.
28) Rafati H. Lavelle EC. Coombes AG. Stolnik S. Holland J. Davis SS. The immune response to a model antigen associated with PLG microparticles prepared using different surfactants. Vaccine 1997, 15(17-18):1888-97.
29) O''Hagan DT. Jeffery H. Davis SS. Long-term antibody responses in mice following subcutaneous immunization with ovalbumin entrapped in biodegradable microparticles. Vaccine 1993, 11(9): 965-9.
30) Uchida T. Goto S. Oral delivery of poly(lactide-co-glycolide) microspheres containing ovalbumin as vaccine formulation: particle size study. Biological & Pharmaceutical Bulletin 1994, 17(9): 1272-6.
31) Flanagan MP. Michael JG. Oral immunization with a Streptococcal pneumoniae polysaccharide conjugate vaccine in enterocoated microparticles induces serum antibodies against type specific polysaccharides. Vaccine 1999, 17(1): 72-81.
32) VanCott JL. Kobayashi T. Yamamoto M. Pillai S. McGhee JR. Kiyono H. Induction of pneumococcal polysaccharide-specific mucosal immune responses by oral immunization. Vaccine 1996, 14(5): 392-8.
33) . Faria AM. Garcia G. Rios MJ. Michalaros CL. Vaz NM. Decrease in susceptibility to oral tolerance induction and occurrence of oral immunization to ovalbumin in 20-38-week-old mice. The effect of interval between oral exposures and rate of antigen intake in the oral immunization. Immunology 1993, 78(1): 147-51.
34) Elson CO. Ealding W. Lefkowitz J. A lavage technique allowing repeated measurement of IgA antibody in mouse intestinal secretions. Journal of Immunological Methods 1984, 67(1): 101-8.
35) Katz JM. Lu X. Young SA. Galphin JC. Adjuvant activity of the heat-labile enterotoxin from enterotoxigenic Escherichia coli for oral administration of inactivated influenza virus vaccine. Journal of Infectious Diseases 1997, 175(2): 352-63.
36) Tabata Y. Inoue Y. Ikada Y. Size effect on systemic and mucosal immune responses induced by oral administration of biodegradable microspheres. Vaccine 1996, 14(17-18): 1677-85.
37) Konradsen HB. Sorensen UB. Henrichsen J. A modified enzyme-linked immunosorbent assay for measuring type-specific anti-pneumococcal capsular polysaccharide antibodies. Journal of Immunological Methods 1993, 164(1): 13-20.
38) Dinarello CA. Interleukin-1 and interleukin-1 antagonism. Blood 1991, 77(8): 1627-52.
39) Siegel JP. Sharon M. Smith PL. Leonard WJ. The IL-2 receptor beta chain (p70): role in mediating signals for LAK, NK, and proliferative activities. Science1987, 238(4823): 75-8.
40) Coffman RL. Shrader B. Carty J. Mosmann TR. Bond MW. A mouse T cell product that preferentially enhances IgA production. I. Biologic characterization. Journal of Immunology 1987, 139(11): 3685-90.
41) Harriman GR. Kunimoto DY. Elliott JF. Paetkau V. Strober W. The role of IL-5 in IgA B cell differentiation. Journal of Immunology 1988, 140(9): 3033-9.
42) Beagley KW. Eldridge JH. Lee F. Kiyono H. Everson MP. Koopman WJ. Hirano T. Kishimoto T. McGhee JR. Interleukins and IgA synthesis. Human and murine interleukin 6 induce high rate IgA secretion in IgA-committed B cells. Journal of Experimental Medicine 1989, 169(6): 2133-48.
43) Abbas AK. Murphy KM. Sher A. Functional diversity of helper T lymphocytes. Nature 1996, 383(6603): 787-93.
44) . Stavnezer J. Regulation of antibody production and class switching by TGF-beta. Journal of Immunology 1995, 155(4): 1647-51.
45) Fan JY. Boyce CS. Cuff CF. T-Helper 1 and T-helper 2 cytokine responses in gut-associated lymphoid tissue following enteric reovirus infection. Cellular Immunology 1998, 188(1): 55-63.
46) Belyakov IM. Derby MA. Ahlers JD. Kelsall BL. Earl P. Moss B. Strober W. Berzofsky JA. Mucosal immunization with HIV-1 peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes and protective immunity in mice against intrarectal recombinant HIV-vaccinia challenge. Proceedings of the National Academy of Sciences of the United States of America 1998, 95(4): 1709-14.
47) Kaiserlian D. Epithelial cells in antigen. Sampling and presentation in mucosal tissues. Current Topics in Microbiology & Immunology 1999, 236: 55-78.
48) Mor TS. Gomez-Lim MA. Palmer KE. Perspective: edible vaccines--a concept coming of age. Trends in Microbiology 1998, 6(11): 449-53.
49) Neutra MR. M cells in antigen sampling in mucosal tissues. Current Topics in Microbiology & Immunology 1999, 236: 17-32.
50) Damge C. Aprahamian M. Marchais H. Benoit JP. Pinget M. Intestinal absorption of PLAGA microspheres in the rat. Journal of Anatomy 1996, 189 ( Pt 3): 491-501.
51) Nono I. Ohno N. Masuda A. Oikawa S. Yadomae T. Oxidative degradation of an antitumor (1-3)-beta-D-glucan, grifolan. Journal of Pharmacobio-Dynamics 1991, 14(1): 9-19.
52) O''Hara Y. [Fate of lentinan (antitumor polysaccharide) I : - fate of lentinan in mice, rats, and dogs (author''s transl)]. Journal of Toxicological Sciences 1980, 5 Suppl: 59-72.
53) Suda M. Ohno N. Adachi Y. Yadomae T. Tissue distribution of intraperitoneally administered (1-->3)-beta-D-glucan (SSG), a highly branched antitumor glucan, in mice. Journal of Pharmacobio-Dynamics 1992, 15(8): 417-26.
54) Suda M. Ohno N. Hashimoto T. Koizumi K. Adachi Y. Yadomae T. Kupffer cells play important roles in the metabolic degradation of a soluble anti-tumor (1-->3)-beta-D-glucan, SSG, in mice. FEMS Immunology & Medical Microbiology 1996, 15(2-3): 93-100.
55) Eldridge JH. Gilley RM. Staas JK. Moldoveanu Z. Meulbroek JA. Tice TR. Biodegradable microspheres: vaccine delivery system for oral immunization. Current Topics in Microbiology & Immunology 1989, 146: 59-66.
56) Faria AM. Weiner HL. Oral tolerance: mechanisms and therapeutic applications. Advances in Immunology 1999, 73: 153-264.
57) Scicchitano R. Biennenstock J. Stanisz AM. In vivo immunomodulation by the neuropeptide substance P. Immunology 1988, 63(4): 733-5.
58) Pascual DW. Xu-Amano JC. Kiyono H. McGhee JR. Bost KL. Substance P acts directly upon cloned B lymphoma cells to enhance IgA and IgM production. Journal of Immunology 1991, 146(7): 2130-6.
59) Stanisz AM. Befus D. Bienenstock J. Differential effects of vasoactive intestinal peptide, substance P, and somatostatin on immunoglobulin synthesis and proliferations by lymphocytes from Peyer''s patches, mesenteric lymph nodes, and spleen. Journal of Immunology 1986, 136(1): 152-6.
60) Boirivant M. Fais S. Annibale B. Agostini D. Delle Fave G. Pallone F. Vasoactive intestinal polypeptide modulates the in vitro immunoglobulin A production by intestinal lamina propria lymphocytes. Gastroenterology 1994, 106(3): 576-82.
61) Annibale B. Fais S. Boirivant M. Delle Fave G. Pallone F. Effects of high in vivo levels of vasoactive intestinal polypeptide on function of circulating lymphocytes in humans. Gastroenterology 1990, 98(6): 1693-8.
62) Liles WC. Van Voorhis WC. Review: nomenclature and biologic significance of cytokines involved in inflammation and the host immune response. Journal of Infectious Diseases 1995, 172(6): 1573-80.
63) Morinaga H. Tazawa K. Tagoh H. Muraguchi A. Fujimaki M. An in vivo study of hepatic and splenic interleukin-1 beta mRNA expression following oral PSK or LEM administration. Japanese Journal of Cancer Research 1994, 85(12): 1298-303.
64) Pozo D. Delgado M. Martinez M. Guerrero JM. Leceta J. Gomariz RP. Calvo JR. Immunobiology of vasoactive intestinal peptide (VIP). Immunology Today 2000, 21(1): 7-11.
65) Strober W. Interactions between epithelial cells and immune cells in the intestine. Annals of the New York Academy of Sciences 1998, 859: 37-45.