摘要
山藥為中國及亞洲國家廣泛使用的藥用植物，具有補中益氣、不飢延年功效。目前仍無研究證實口服山藥能促進免疫調節功能。本實驗首先便觀察口服山藥塊莖去除澱粉後所得多糖成份對免疫反應的影響。我們證實，在系統免疫方面，口服山藥多糖能明顯促進脾臟自然殺手細胞毒殺作用、脾臟細胞對裂殖原Con A反應的能力，及骨髓細胞對顆粒球/單核球幹細胞生長因子刺激增生的反應。
在黏膜免疫方面，我們利用兩種不同抗原：白蛋白及肺炎雙球莢膜多糖疫苗，以口服方式引發小鼠的抗體免疫反應，同時將山藥多糖加入小鼠日常飲水中作為免疫調節劑。結果證實，山藥多糖可以引起肺炎雙球莢膜多糖疫苗專一性的血清IgG及黏膜IgA抗體反應。並且能夠突破口服耐受性限制，引起口服水溶性白蛋白的小鼠，血清IgG及黏膜IgA的抗體免疫反應。
為了解山藥多糖在黏膜免疫調節機制，我們進一步觀察腸道細胞激素反應，包括腸道上皮淋巴細胞、固有層及Peyer’s patch等組織。在固有層及Peyer’s patch中，輔助性T細胞第一型細胞激素，IFN-；第二型細胞激素IL-4、IL-6；以及第三型細胞激素TGF-，均因口服山藥多糖而被大量刺激表現。這或許可以解釋山藥多糖對腸道免疫調節的作用。另外，山藥多糖能促進腸道上皮細胞中，具有抵抗微生物能力的小分子蛋白質cryptdin (defensin family)基因大量表現。因此，由細胞激素表現的結果顯示，山藥可能透過細胞激素活性的調控，促進黏膜免疫反應，達到免疫調節的作用。
山藥所含的類固醇已被應用於合成人體所需的多種類固醇產品。實驗初步證實口服山藥甲醇粗萃物對於系統免疫反應沒有顯著的生物活性。但是在體外實驗(in vitro)中，山藥甲醇粗萃物對於腦神經及骨髓中的祖原細胞則具有促進細胞增生及形態改變的活性。
骨質疏鬆症造成的原因包括骨形成與再吸收的速率失去平衡。成骨細胞負責骨形成，鹼性磷酸為成骨細胞活性的標記。山藥甲醇粗萃物能刺激骨細胞增生及分化，以補充骨中的造骨祖原細胞及促進造骨細胞的成熟。
幹細胞在發育過程中主要參與在組織更新，以確保器官長久生存。源自成體特定組織的幹細胞，目前發現具有替代多種身體組織的潛力。山藥甲醇粗萃物或許因此能藉由促進細胞再生的活性，預防或治療一些與老化相關的疾病，如神經退化性疾病及骨質疏鬆症。
在本研究中，我們了解山藥多糖在系統及黏膜免疫上確實具有免疫調節作用。且山藥低分子的甲醇粗萃物具有促進腦神經及骨髓祖原細胞生長及分化的細胞再生能力。

Abstract
Dioscorea spp., a medical herb, has been widely used as a remedy for promotion of health and longevity in China and other Asian countries, but no scientific evidences of their immune-potentiation effects by oral administration have been documented. In our study, immune responses of non-starch polysaccharide components of tubers from Dioscorea spp. by oral administration were examined. In systemic immunity, the cytotoxic activity of natural killers, and the proliferative activity of ConA-induced splenocytes, the proliferative activity of GM-CSF-stimulated bone marrow cells on Dioscorea polysaccharides diet were significantly enhanced.
In mucosal immunity, we used two different antigens： ovalbumin and Pneumovax 23 vaccine to induce humoral immune responses in mice by gastric intubation. Additionally, Dioscorea polysaccharides were supplemented in daily drink for mice as an immunomodulator. For an immunomodulator, a feed of Dioscorea polysaccharides not only abrogated the induction of oral tolerance stimulated by ovalumin feeding in mice but also induced the ovalbumin and Pneumovax-specific serum IgG and mucosal IgA antibody responses.
We attempted to clarify the mechanism of the mucosal immunomodulation of Dioscorea polysaccharides on cytokine responses. Cytokine production by intestinal epithilium lymphocytes, lamina propria and Peyer’s patch of mice orally administrated with Dioscorea polysaccharides was examined. Weak Th1 cytokine IFN-gamma, strong Th2 cytokine IL-4, IL-6 and TGF mRNA expressions were induced. Innate anti-microbial peptide cryptdin (defensin family) mRNA production by intestinal epithilium lymphocytes was also induced. Taken together, our study demonstrated that Dioscorea polysaccharides have differential effects on the expression of various cytokines, which likely contribute to its immunomodulatory activity.
Dioscorea steroid extract is used in commercial steroid synthesis and consumed by people. In our study, methanol extracts of Dioscorea had no obvious effects in systemic immune responses. But when we investigated the effect of methanol extracts of Dioscorea on the progenitor cells, we found that methanol extract of Dioscorea added to neuron progenitor cells and bone marrow cell cultures induced stem cell proliferation and morphological changes.
Bone loss in postmenopausal osteoporosis is mainly due to decreased bone formation or increased bone resorption. Bone formation is mediated by osteoblasts and alkaline phosphatase is a biological marker of osteoblast activity. We proved that methanol extract of Dioscorea stimulation of bone cell proliferation and differentiation, possibly to promote the recruitment and maturation of primitive osteogenic precursors present of bone and marrow.
Stem cells may arise late in development, to act principally in tissue renewal, thus ensuring an organism''''''''s long-term survival. Tissue-specific adult stem cells have the potential to contribute to replenishment of multiple adult tissues. Methanol extracts of Dioscorea may improve cell regeneration to prevent or cure several age-related diseases such as neuron degeneration diseases and osteoporosis.
In this study, we demonstrated that polysaccharides of Dioscorea spp. did have their effects on immunomodulation in both systemic and mucoal immunity. And methanol crude extract of Dioscorea spp. contains bioacitive compounds capable of stimulating the proliferation and differentiation of progenitor cells in brain and bone marrow, in related with their cell regeneration function.

目錄
目錄……………………………………………………………………… I
圖次…………………………………………………………………… II
表次…………………………………………………………………… IV
縮寫表………………………………………………………………… VI
中文摘要……………………………………………………………… 1
英文摘要……………………………………………………………… 3
緒論…………………………………………………………………… 6
試藥及器材…………………………………………………………… 14
方法…………………………………………………………………… 21
結果…………………………………………………………………… 36
討論…………………………………………………………………… 43
參考文獻……………………………………………………………… 56
圖表說明……………………………………………………………… 67
附圖…………………………………………………………………… 104
圖次
圖1：Purification of mGM-CSF ……………………………………………… 67
圖2：Effect of Dioscorea polysaccharides on mitogenic responses of Con A- stimulated
splenocytes in C3H mice………………………………………………………… 68
圖3：Effect of Dioscorea polysaccharides on natural killer cell mediated cytotoxicity
in C3H mice……………………………………………………………………… 69
圖4：Effect of Dioscorea polysaccharides on the proliferation activity of bone marrow
cells in C3H mice………………………………………………………………… 70
圖5：Effects of Pneumovax 23 vaccine specific mucosal IgA induced by oral
immunization and long term feeding with Dioscorea polysaccharides as an
immunomodulator on 20 wk C57BL/6 mice. …………..................……..… 71
圖6：Effects of Pneumovax 23 vaccine specific mucosal IgA induced by oral
immunization and long term feeding with Dioscorea polysaccharides as an
immunomodulator on 10 wk C57BL/6 mice.…………………………….. 72
圖7：Effects of ovalbumin specific mucosal IgA antibody induced by oral
immunization and long term feeding with Dioscorea polysaccharides as an
immunomodulator on 20wk C57BL/6 mice……………………………… 73
圖8：Effects of ovalbumin specific mucsoal IgA antibody induced by oral
immunization and long term feeding with Dioscorea polysaccharides as an
immunomodulator on 20wks C3H mice .……………………….……… 74
圖9：Effects of Pneumovax 23 vaccine specific serum antibodies induced by oral
immunization and long term feeding with Dioscorea polysaccharides as an
immunomodulator on 10 wk C57BL/6 mice…………………………………….. 75
圖10：Effects of ovalbumin specific serum IgG and IgM antibody induced by oral
immunization and long term feeding with Dioscorea polysaccharides as an
immunomodulator on 20 wk C57BL/6 mice……………………….……. 76
圖11：Effects of ovalbumin specific serum IgG and IgM antibody induced by oral
immunization and long term feeding with Dioscorea polysaccharides as an
immunomodulator on 20wk C3H mice…………….……………………... 77
圖12：Polymerase chain reaction analysis of cytokine-specific mRNA in Peyer’s patch
isolated from mice fed with Dioscorea polysaccharides………………………… 78
圖13：Polymerase chain reaction analysis of cytokine-specific mRNA in lamina propria
isolated from mice fed with Dioscorea polysaccharides………………………… 79
圖14：Polymerase chain reaction analysis of cryptdin mRNA in enterocyte isolated from
mice fed with Dioscorea polysaccharides……………………………...………… 80
圖15：Effects of methanol extracts of Dioscorea on GM-SCF-stimulated bone marrow
cells in C3H mice……………………………………………...………………… 81
圖16：Effects of methanol extracts of Dioscorea on mitogenic responses of Con A-
stimulated splenocytes in C3H mice……………………………………...………. 82
圖17：Effects of methanol extracts of Dioscorea on natural killer cell- mediated
cytotoxicity in C3H mice.……………………………............………...……….… 83
圖18：Polymerase chain reaction analysis of cytokine-specific mRNA in lamina propria
isolated from mice fed with MeOH extracts of Dioscorea.……………………..... 84
圖19：Effects of different partition fractions of methanol extracts of Dioscorea
and DHEA on mitogenic responses of osteogenic cell in vitro........... 85
圖20：Orally administration effects of methanol extracts of Dioscorea on mitogenic
responses of osteogenic cell in C57BL/6 mice...................................................... 86
圖21：Effects of different partition fractions of methanol extracts of Dioscorea
on alkaline phosphatase activity in osteoblasts cultures in vitro……………… 87
圖22：Orally administration effects of methanol extracts of Dioscorea on on
alkaline phosphatase activity in osteoblasts cultures in C57BL/6 mice..………… 88
圖23：Effects of methanol extracts of Dioscorea on morphological changes of murine
osteoblast cells.…...………….….….….….……..…….……………………… 89
圖24：Effects of methanol extracts of Dioscorea on morphological changes of
the primary cultured bone marrow cells from P5 mice…………………… 90
圖25：Effects of DHEA on morphological changes of the primary cultured
bone marrow cells from P5 mice............................................................................. 91
圖26：Effects of different partition fractions of methanol extracts of Dioscorea
and DHEA on proliferation responses of bone marrow cells in C3H mice. 92
圖27：Effects of methanol extracts of Dioscorea on morphological changes of
the primary cultured bone marrow cells from 8wk mice….………..…… 93
圖28：Effect of DHEA on morphological changes of the primary cultured bone
marrow cells from 8wk mice...........................................….………...................… 94
圖29：Effects of methanol extracts of Dioscorea on morphological changes of
secondary passaged P1 brain progenitor cells…………..…..……..…… 95
表次
表1：List of the weights and yields of partition fractions of Dioscorea spp…………… 96
表2：List of weights and recoveries of rmGM-CSF purification……………………… 97
表3：Effects of different partition fractions of methanol extracts of Dioscorea spp. on
the EGF-stimulated proliferation and morphological change of primary cultured
bone marrow cells from 20wk mice……………………………..………………… 98
表4：Effects of DHEA on the EGF-stimulated proliferation and morphological change
of primary cultured bone marrow cells from 8wk mice…………………………… 99
表5：Effects of methanol extracts of Dioscorea spp. on the GM-CSF-stimulated
proliferation and morphological change of primary cultured bone marrow cells
from 20wk mice……………………….…………………………….……..……… 100
表6：Effects of DHEA and different partition fractions of methanol extracts of
Dioscorea spp. on the GM-CSF-stimulated proliferation and morphological
change of primary cultured bone marrow cells from 8wk mice…………………... 101
表7：Effects of methanol extracts of Dioscorea spp. on the proliferation and
morphological change of secondary passage P1 progenitor cells from mice brain.. 102
表8：Effects of different partition fractions of methanol extracts of Dioscorea spp. on
the proliferation and morphological change of secondary passage P1 progenitor
cells from mice brain.......….........….........….........………………………………. 103

參考文獻
1. Liu S.Y. Wang J.Y. Shyu Y.T. Song L.M. Studies on Yams (Dioscorea spp.) in Taiwan. J.Chin.Med. 6: 111-126, 1995.
2. 後漢作品，神農本草經，佚。孫星衍、孫馮翼重輯：神農本草經(1799) ，自由出版社重印，台北 1969；卷1：18。
3. 明，李時珍：本草綱目(1593)，宏業書局印行，台北 1979；中冊119-120.
4. 許鴻源：中藥材之研究，新醫藥出版社，台北 1980: 180。
5. Shuying H. Tong Z. Shuru W. Study on chemistry and antioxidation activity of water-soluble polysaccharides of rhizoma Dioscoreae Oppositae. J. Chin. Phar. Uni. 25(6): 369-372, 1994.
6. Hikino H. Konno C. Takahashi M. Murakami M. Kato Y. Karikura M. Hayashi T. Isolation and hypoglycemic activity of dioscorans A, B, C, D, E, and F; glycans of Dioscorea japonica rhizophors. Planta Medica. (3): 168-171, 1986.
7. Ogra, P.L. (ed.): Mucosal immunology. San Diago: Academic press, 1999.
8. Burgess AW. Camakaris J. Metcalf D. Purification and properties of colony-stimulating factor from mouse lung-conditioned medium. Journal of Biological Chemistry. 252(6):1998-2003, 1977
9. Trowbridge IS. Mitogenic properties of lectin and its chemical dervatives. Proc Natl Acad Sci U S A. 70(12): 3650-3654, 1973.
10. Stobo JD. Phytohemagglutin and concanavalin A: probes for murine ''''''''T'''''''' cell activivation and differentiation. Transplantation Reviews. 11:60-86, 1972.
11. Kirkwood JM. Ernstoff MS. Interferons in the treatment of human cancer. J Clin Oncol. 2(4): 336-352, 1984.
12. J Clin Oncol. Ouellette, A.J. and Selsted, M.E. Paneth cell defensins: endogenous peptide components of intestinal host defense. FASEB Journal 10(11): 1280-1289, 1996.
13. Ouellette AJ. Greco RM. James M. Frederick D. Naftilan J. Fallon JT. Developmental regulation of cryptdin, a corticostatin/defensin precursor mRNA in mouse small intestinal crypt epithelium. Journal of Cell Biology. 108(5): 1687-1695, 1989.
14. Ouellette A.J. Paneth cells and innate immunity in the crypt microenvironment. Gastroenterology 113(5): 1779-1884, 1997.
15. Kagan B.L. Ganz T. Lehrer R.I. Defensins: a family of antimicrobial and cytotoxic peptides. Toxicology 87(1-3): 131-149, 1994.
16. Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. Enteric defensins: antibiotic peptide components of intestinal host defense. Journal of Cell Biology 118(4): 929-936, 1992.
17. Qu XD. Lloyd KC. Walsh JH. Lehrer RI. Secretion of type II phospholipase A2 and cryptdin by rat small intestinal Paneth cells. Infection & Immunity. 64(12): 5161-5165, 1996.
18. Ouellette AJ. Hsieh MM. Nosek MT. Cano-Gauci DF. Huttner KM. Buick RN. Selsted ME. Mouse Paneth cell defensins: primary structures and antibacterial activities of numerous cryptdin isoforms. Infection & Immunity. 62(11): 5040-5047, 1994.
19. Husby S. Normal immune responses to ingested foods. Journal of Pediatric Gastroenterology & Nutrition. 30: S13-19. 2000.
20. Mayer L. Mucosal immunity and gastrointestinal antigen processing. Journal of Pediatric Gastroenterology & Nutrition. 30: S4-12, 2000.
21. McGhee J.R. Kiyono H. The mucosal immune system. In: Anonymous Fundamental immunology, Paul W.E. (ed.) Philadelphia: Lippincott- Ravan, 1999, pp 909-945.
22. Kantele A. Westerholm M. Kantele JM. Makela PH. Savilahti E. Homing potentials of circulating antibody-secreting cells after administration of oral or parenteral protein or polysaccharide vaccine in humans. Vaccine. 17(3): 229-236, 1999.
23. MacDonald TT. Effector and regulatory lymphoid cells and cytokines in mucosal sites. Current Topics in Microbiology & Immunology. 236: 113-135, 1999.
24. Janoff EN. Fasching C. Orenstein JM. Rubins JB. Opstad NL. Dalmasso AP. Killing of Streptococcus pneumoniae by capsular polysaccharide specific polymeric IgA, complement, and phagocytes. Journal of Clinical Investigation. 104(8): 1139-1147, 1999.
25. Boyaka PN. Marinaro M. Vancott JL. Takahashi I. Fujihashi K. Yamamoto M. van Ginkel FW. Jackson RJ. Kiyono H. McGhee JR. Strategies for mucosal vaccine development. American Journal of Tropical Medicine & Hygiene. 60(4): 35-45, 1999.
26. Garg M. Luo W. Kaplan AM. Bondada S. Cellular basis of decreased immune responses to pneumococcal vaccines in aged mice. Infection & Immunity. 64(11): 4456-4462, 1996.
27. Van den Dobbelsteen GP. Van Rees EP. Mucosal immune responses to pneumococcal polysaccharides: implications for vaccination. Trends in Microbiology. 3(4): 155-159, 1995.
28. Flanagan MP. Michael JG. Oral immunization with a Streptococcal pneumoniae polysaccharide conjugate vaccine in enterocoated microparticles induces serum antibodies against type specific polysaccharides. Vaccine. 17(1): 72-81, 1999.
29. VanCott JL. Kobayashi T. Yamamoto M. Pillai S. McGhee J.R. Kiyono H. Induction of pneumococcal polysaccharide-specific mucosal immune responses by oral immunization. Vaccine, 14: 392-398, 1996.
30. Buchanan RM. Arulanandam BP. Metzger DW. IL-12 enhances antibody responses to T-independent polysaccharide vaccines in the absence of T and NK cells. Journal of Immunology. 161(10): 5525-5533, 1998.
31. Yamamoto M. McDaniel LS. Kawabata K. Briles DE. Jackson RJ. McGhee JR. Kiyono H. Oral immunization with PspA elicits protective humoral immunity against Streptococcus pneumoniae infection. Infection & Immunity. 65(2): 640-644, 1997.
32. Araghiniknam M. Chung S. Nelson-White T. Eskelson C. Watson R.R. Antioxidant activity of dioscorea and dehydro-epiandrosterone (DHEA) in older humans. Life Sciences, 59: 147-157, 1996.
33. Li C. Yu B. Liu M. Hui Y. Synthesis of diosgenyl alpha-L-rhamno-pyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->3)]-beta-D-glucopyranoside (gracillin) and related saponins. Carbohydrate. Research, 306: 189-195, 1998.
34. Aradhana. Rao AR. Kale RK. Diosgenin--a growth stimulator of mammary gland of ovariectomized mouse. Indian Journal of Experimental Biology. 30(5): 367-370, 1992.
35. Van der Kooy D. Weiss S. Why stem cells? Science. 287(5457): 1439-1441, 2000.
36. Kuhn HG. Dickinson-Anson H. Gage FH. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. Journal of Neuroscience. 16(6): 2027-2033, 1996.
37. Weissman L. Stem cells: units of development, units of regeneration, and units in evolution. Cell. 100(1): 157-168, 2000.
38. Flax JD. Aurora S. Yang C. Simonin C. Wills AM. Billinghurst LL. Jendoubi M. Sidman RL. Wolfe JH. Kim SU. Snyder EY. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nature Biotechnology. 16(11): 1033-1039, 1998.
39. Gritti A. Cova L. Parati EA. Galli R. Vescovi AL. Basic fibroblast growth factor supports the proliferation of epidermal growth factor-generated neuronal precursor cells of the adult mouse CNS. Neuroscience Letters. 185(3): 151-154, 1995.
40. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 276(5309): 71-74, 1997.
41. Martin I. Muraglia A. Campanile G. Cancedda R. Quarto R. Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow. Endocrinology. 138(10): 4456-4462, 1997.
42. Woodbury D. Schwarz EJ. Prockop DJ. Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. Journal of Neuroscience Research. 61(4): 364-370, 2000.
43. Scheven BA. Milne JS. Dehydroepiandrosterone (DHEA) and DHEA-S interact with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to stimulate human osteoblastic cell differentiation. Life Sciences. 62(1): 59-68, 1998.
44. Cheng SL. Yang JW. Rifas L. Zhang SF. Avioli LV. Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology. 134(1): 277-286, 1994.
45. QU Q. PH M. Kapanen A. Dahllund J. Salo J. Vaananen H.K. Harkonen P. Estrogen enhances differentiation on osteoblasts in mouse bone marrow culture. Bone 22: 201-209, 1998.
46. Schrimsher J.L. Rose K. Simona M.G. Wingfield P. Characterization of human and mouse granulocyte-macrophage-colony-stimulating factors derived from Escherichia coli. Biochemical Journal, 247: 195-199, 1987.
47. DeLamarter J.F. Mermod J.J. Liang C.M. Eliason J.F. Thatcher D.R. Recombinant murine GM-CSF from E. coli has biological activity and is neutralized by a specific antiserum. EMBO Journal, 4: 2575-2581, 1985.
48. Metcalf D. Burgess A.W. Johnson G.R. Nicola N.A. Nice E.C. DeLamarter J. Thatcher D.R. Mermod J.J. In vitro actions on hemopoietic cells of recombinant murine GM-CSF purified after production in Escherichia coli: comparison with purified native GM-CSF. Journal of Cellular Physiology, 128: 421-431, 1986.
49. Huttner K.M. Selsted M.E. Ouellette A.J. Structure and diversity of the murine cryptdin gene family. Genomics 19(3): 448-553, 1994.
50. Liu, F., Ooi, V.E., and Fung, M.C. Analysis of immunomodulating cytokine mRNAs in the mouse induced by mushroom polysaccharides. Life Sciences, 64: 1005-1011, 1999.
51. Fan J.Y. Boyce C.S. Cuff C.F. T-Helper 1 and T-helper 2 cytokine responses in gut-associated lymphoid tissue following enteric reovirus infection. Cellular Immunology, 188: 55-63, 1998.
52. Cowdery J.S. McKiernan F.E. Analysis of T cell and B cell function in Peyer''''''''s patch and lamina propria of New Zealand Black and DBA/2 mice. Journal of Immunology, 136: 4070-4074. 1986.
53. DeWitt RC. Wu Y. Renegar KB. King BK. Li J. Kudsk KA. Bombesin recovers gut-associated lymphoid tissue and preserves immunity to bacterial pneumonia in mice receiving total parenteral nutrition. Annals of Surgery. 231(1): 1-8, 2000.
54. Uchida T. Goto S. Oral delivery of poly (lactide-co-glycolide) microspheres containing ovalbumin as vaccine formulation: particle size study. Biological & Pharmaceutical Bulletin. 17(9): 1272-1276, 1994.
55. Elson C.O. Ealding W. Lefkowitz J. A lavage technique allowing repeated measurement of IgA antibody in mouse intestinal secretions. Journal of immunological methods, 67: 101-108. 1984.
56. Katz J.M. Lu X. Young S.A. Galphin J.C. Adjuvant activity of the heat-labile enterotoxin from enterotoxigenic Escherichia coli for oral administration of inactivated Influenza virus vaccine. Journal of infectious disease, 175: 352-363, 1997.
57. 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. 15(17-18): 1888-1897, 1997.
58. Konradsen, H.B., Sorensen, U.B. and Henrichsen, J. A modified enzyme-linked immunosorbent assay for measuring type-specific anti-pneumococcal capsular polysaccharide antibodies. Journal of immunological methods, 164: 13-20. 1993.
59. Sabokbar A. Millett PJ. Myer B. Rushton N. A rapid, quantitative assay for measuring alkaline phosphatase activity in osteoblastic cells in vitro. Bone & Mineral. 27(1): 57-67, 1994.
60. Hang Y.Y. Determination of the content of main constituents and pharmacological experiments of Dioscorea japonica in China. Journal of Plant resource and environment, 5: 5-8, 1996.
61. Haq TA. Mason HS. Clements JD. Arntzen CJ. Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science. 268(5211): 714-716, 1995.
62. Schaefer W. Goerz A. Kahl G. T DNA integration and expression in a monocot crop plant after induction of agrobacterium. Nature 327(6122): 529-532, 1987.
63. Strober W. Kelsall B. Marth T. Oral tolerance. Journal of Clinical Immunology. 18(1): 1-30, 1998.
64. Elson CO. Ealding W. Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin. Journal of Immunology. 132(6): 2736-2741, 1984.
65. 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. 78(1): 147-151, 1993.
66. Faria AM. Weiner HL. Oral tolerance: mechanisms and therapeutic applications. Advances in Immunology. 73:153-264, 1999.
67. Romero-Steiner S. Musher DM. Cetron MS. Pais LB. Groover JE. Fiore AE. Plikaytis BD. Carlone GM. Reduction in functional antibody activity against Streptococcus pneumoniae in vaccinated elderly individuals highly correlates with decreased IgG antibody avidity. Clinical Infectious Diseases. 29(2): 281-288, 1999.
68. Husband AJ. Bao S. Beagley KW. Analysis of the mucosal microenvironment: factors determining successful responses to mucosal vaccines. Veterinary Immunology & Immunopathology. 72(1-2): 135-142, 1999.
69. Strober W. Interactions between epithelial cells and immune cells in the intestine. Annals of the New York Academy of Sciences. 859:37-45, 1998.
70. 潘宜欣。(2000) 靈芝多糖於腸道免疫系統作用的研究 國立陽明大學生物藥學研究所碩士論文。
71. Xu-Amano J. Jackson RJ. Fujihashi K. Kiyono H. Staats HF. McGhee JR. Helper Th1 and Th2 cell responses following mucosal or systemic immunization with cholera toxin. Vaccine. 12(10): 903-911, 1994.
72. Sieff CA. Emerson SG. Donahue RE. Nathan DG. Wang EA. Wong GG. Clark SC. Human recombinant granulocyte- macrophage colony- stimulating factor: a multilineage hematopoietin. Science. 230(4730): 1171-1173, 1985.
73. 李樹英 五種山藥對小鼠免疫功能影響的比較研究 河南中醫, 12: 23-24, 1992.
74. 周可范 山藥的研究概況 中草藥, 24: 158-160, 1993.
75. 苗明三 懷山藥對免疫功能的影響 河南中醫, 16: 349-350, 1996.
76. Sakushima J. Nose M. Ogihara Y. Effect of hachimi-jio-gan on immunoglobulin A producing cells in Peyer''''''''s patch by oral administration. Biological & Pharmaceutical Bulletin. 20(11): 1175-1177, 1997.
77. Kumazawa Y. Mizunoe K. Otsuka Y. Immunostimulating polysaccharide separated from hot water extract of Angelica acutiloba Kitagawa (Yamato tohki). Immunology. 47(1): 75-83, 1982.
78. Han SB. Kim YH. Lee CW. Park SM. Lee HY. Ahn KS. Kim IH. Kim HM. Characteristic immunostimulation by angelan isolated from Angelica gigas Nakai. Immunopharmacology. 40(1): 39-48, 1998.
79. Tsukamoto T., Ueno Y, Ohta Z. Glycosides of Diosocrea tokoro I. Dioscin, dioscoreasapotoxin and diosgenin. J. Pharm Soc Jpn 56: 135, 1936.
80. Zava DT. Dollbaum CM. Blen M. Estrogen and progestin bioactivity of foods, herbs, and spices. Experimental Biology & Medicine. 217(3): 369-378, 1998.
81. Evans TG. Judd ME. Dowell T. Poe S. Daynes RA. Araneo BA. The use of oral dehydroepiandrosterone sulfate as an adjuvant in tetanus and influenza vaccination of the elderly. Vaccine. 14(16): 1531-1537, 1996.
82. Caffrey RE. Kapasi ZF. Haley ST. Tew JG. Szakal AK. DHEAS enhances germinal center responses in old mice. Advances in Experimental Medicine & Biology. 355: 225-229, 1994.
83. Ronald, F. Dehydroepiandrosterone in Systemic Lupus Erythe-matosus Rheumatic disease. Clinics of North America. 26: 349-363, 2000.
84. Catalina F. Kumar V. Milewich L. Bennett M. Food restriction-like effects of dehydroepiandrosterone: decreased lymphocyte numbers and functions with increased apoptosis. Experimental Biology & Medicine. 221(4): 326-335, 1999.
85. Qu Q. Harkonen PL. Monkkonen J. Vaananen HK. Conditioned medium of estrogen-treated osteoblasts inhibits osteoclast maturation and function in vitro. Bone. 25(2): 211-215, 1999.
86. Paula Kiberstis, Orla Smith, and Colin Norman. Bone Health in the Balance. Science 289: 1497, 2000.
87. Locklin RM. Williamson MC. Beresford JN. Triffitt JT. Owen ME. In vitro effects of growth factors and dexamethasone on rat marrow stromal cells. Clinical Orthopaedics & Related Research. (313): 27-35, 1995.
88. Nawata H. Tanaka S. Tanaka S. Takayanagi R. Sakai Y. Yanase T. Ikuyama S. Haji M. Aromatase in bone cell: association with osteoporosis in postmenopausal women. Journal of Steroid Biochemistry & Molecular Biology. 53(1-6): 165-174, 1995.
89. Higdon K. Scott A. Tucci M. Benghuzzi H. Tsao A. Puckett A. Cason Z. Hughes J. The use of estrogen, DHEA, and diosgenin in a sustained delivery setting as a novel treatment approach for osteoporosis in the ovariectomized adult rat model. Biomedical Sciences Instrumentation. 37: 281-286, 2001.
90. Martel C. Sourla A. Pelletier G. Labrie C. Fournier M. Picard S. Li S. Stojanovic M. Labrie F. Predominant androgenic component in the stimulatory effect of dehydroepiandrosterone on bone mineral density in the rat. Journal of Endocrinology. 157(3): 433-442, 1998.
91. Ueno K. Katayama T. Miyamoto T. Koshihara Y. Interleukin-4 enhances in vitro mineralization in human osteoblast-like cells. Biochemical & Biophysical Research Communications. 189(3): 1521-1526, 1992.
92. Orwoll E. Ettinger M. Weiss S. Miller P. Kendler D. Graham J. Adami S. Weber K. Lorenc R. Pietschmann P. Vandormael K. Lombardi A. Alendronate for the treatment of osteoporosis in men. New England Journal of Medicine. 343(9): 604-610, 2000.
93. Fisher JE. Rogers MJ. Halasy JM. Luckman SP. Hughes DE. Masarachia PJ. Wesolowski G. Russell RG. Rodan GA. Reszka AA. Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro. Proceedings of the National Academy of Sciences of the United States of America. 96(1): 133-138, 1999.
94. Tushinski RJ. Stanley ER. The regulation of mononuclear phagocyte entry into S phase by the colony stimulating factor CSF-1. Journal of Cellular Physiology. 122(2): 221-228, 1985.
95. Yamada M. Suzu S. Akaiwa E. Wakimoto N. Hatake K. Motoyoshi K. Shimamura S. Properties of primary murine stroma induced by macrophage colony-stimulating factor. Journal of Cellular Physiology. 173(1): 1-9, 1997.
96. Beneytout JL. Nappez C. Leboutet MJ. Malinvaud G. A plant steroid, diosgenin, a new megakaryocytic differentiation inducer of HEL cells. Biochemical & Biophysical Research Communi-cations. 207(1): 398-404, 1995.
97. Reynolds BA. Weiss S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Developmental Biology. 175(1): 1-13, 1996.
98. Bjornson CR. Rietze RL. Reynolds BA. Magli MC. Vescovi AL. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science. 283(5401): 534-537, 1999.
99. Olanow CW. Kordower JH. Freeman TB. Fetal nigral transplantation as a therapy for Parkinson''''''''s disease. Trends in Neurosciences. 19(3): 102-109, 1996.
100. Svendsen CN. Smith AG. New prospects for human stem-cell therapy in the nervous system. Trends in Neurosciences. 22(8): 357-364, 1999.
101. Gage FH. Cell therapy. Nature. 392: 18-24, 1998.
102. Khatim MS. Al-Mahmood HA. Gumaa KA. Modulation of rat pancreatic islet cell replication and insulin release by glibenclamide. FEBS Letters. 177(1): 135-7, 1984.
103. Jackson RA. Hawa MI. Jaspan JB. Sim BM. Disilvio L. Featherbe D. Kurtz AB. Mechanism of metformin action in non-insulin-dependent diabetes. Diabetes. 36(5): 632-640, 1987.