臺灣博碩士論文加值系統

樹突細胞為最有效率抗原呈獻細胞，對於引發免疫反應有著舉足輕重
的影響力。樹突細胞不僅是先天性免疫的一員，也操控著抗原專一性的免
疫反應。先前的文獻中證實桑色素、桑色素結合態代謝物、槲皮素結合態
代謝物等黃酮類化合物具有抑制巨噬細胞活性的功能。然而，這些化合物
是否影響樹突細胞功能目前尚無任何研究報告。我們利用老鼠骨髓衍生之
樹突細胞來評估桑色素、桑色素結合態代謝物、槲皮素結合態代謝物對樹
突細胞細胞表面標記的表現、抗原呈獻的能力及促使T 細胞增生、細胞激
素分泌及誘導T 細胞分化之影響。在樹突細胞培養至第4 天或第6 天時，
分別給予或不給予脂多醣及不同濃度的桑色素(0, 5, 10, 20 µM)、桑色素結合態代謝物(0, 5, 10, 20 µM)、槲皮素結合態代謝物(0, 4, 8, 16 µM)，再刺激2 天後進行分析。結果證實不論是否給予給予脂多醣，在桑色素20 µM、桑色素結合態代謝物20 µM 或槲皮素結合態代謝物16 µM 存在情況下，均顯著降低樹突細胞表面標記的表現及抗原呈獻的能力，且會調節T 細胞分化為Th2。這些黃酮類化合物雖互為結構異構物，但是對老鼠骨髓衍生的樹突細胞影響是一致的。根據我們目前的發現，值得進一步探討桑色素、桑色素結合態代謝物、槲皮素結合態代謝物影響樹突細胞的作用機轉，未來將可以為中藥應用於自體免疫疾病的免疫治療提供更多的資訊。

Dendritic cells (DCs) are potent antigen-presenting cells with a unique ability to induce immune responses. DCs are not only critical for the induction of primary immune responses, but also important for the control of adaptive immune responses.
Previous studies showed that some flavonoids inhibit macrophage activities. However,there is not any biological research about the effects of the flavonoids such as morin,morin conjugated metabolites and quercetin conjugated metabolites on the functions of DCs. Here, we study and compare the effects of morin, morin conjugated metabolites and quercetin conjugated metabolites on mice bone marrow-derived DCs including the surface marker expression, antigen presentation ability, cytokine secretion and the induction of T cells differentiation. At day 4 or day 6,lipopolysaccharide(LPS) was added with different concentrations of morin (0, 5, 10,20 μM), morin conjugated metabolites(0, 5, 10, 20 μM), or quercetin conjugated metabolites(0, 4, 8, 16 μM), respectively. After 2 days the characteristics of DCs were analyzed. The results demonstrated that in the presence or absence of LPS, 20
μM morin, 20 μM morin conjugated metabolites, or 16 μM quercetin conjugated metabolites, could significantly inhibit DCs surface marker expression, and antigen presentation ability. Meanwhile, treated DCs promote T cells differentiate to Th2 cells.Although morin, morin conjugated metabolites, quercetin conjugated metabolites are isomeric flavonoid, their effects on bone marrow-derived DCs are similar. Based on
our findings, we could further study the mechanisms of the immunological effects of morin, morin conjugated metabolites, and quercetin conjugated metabolites on DCs.This study will provide more information about the application of Chinese herbs on the immunotherapy of autoimmune diseases.

標題……………………………………………………………………………………i
誌謝……………………………………………………………………………………ii
中文摘要………………………………………………………………………………iii
英文摘要………………………………………………………………………………Ⅳ
縮寫表…………………………………………………………………………………Ⅴ
目錄……………………………………………………………………………………Ⅵ
第一章緒論
第一節樹突細胞(dendritic cells, DCs)…………………………………………….. 1
第二節桑色素及其代謝物PD-02…………………………………………………..3
第三節槲皮素之結合態代謝物(PD-01)…………………………………………..4
第四節研究動機與目的…………………………………………………………..6
第五節實驗設計…………………………………………………………………..7
第二章實驗方法………………………………………………………………….8
第三章實驗結果
第一節培養骨髓衍生之樹突細胞…………………………………………….. 14
第二節評估脂多醣對樹突細胞其細胞表面標記的影響…………………………14
第三節評估桑色素對於樹突細胞其細胞表面標記的影響………………………15
第四節評估桑色素結合態代謝物(PD-02)對於樹突細胞其細胞表面標記
的影響……………………………………………………………………17
第五節評估槲皮素結合態代謝物(PD-01)對於樹突細胞其細胞表面標記
的影響……………………………………………………………………20
第六節評估桑色素對於樹突細胞釋放細胞激素之影響…………………………22
第七節評估桑色素結合態代謝物(PD-02)對於樹突細胞釋放細胞激素之影響…23
第八節評估槲皮素結合態代謝物(PD-01)對於樹突細胞釋放細胞激素之影響…24
第九節評估桑色素對於樹突細胞其抗原呈獻作用之影響……………………25
第十節評估桑色素結合態代謝物(PD-02)對於樹突細胞其抗原呈獻作用
之影響………………………………………………………………………26
第十一節評估槲皮素結合態代謝物(PD-01)對於樹突細胞其抗原呈獻作用
之影響……………………………………………………………………27
第十二節評估經由桑色素處理的樹突細胞對於淋巴球釋放細胞激素之影響…28
第十三節評估經由桑色素結合態代謝物(PD-02)處理的樹突細胞對於淋巴
球釋放細胞激素之影響………………………………………………30
第十四節評估經由槲皮素結合態代謝物(PD-01)處理的樹突細胞對於淋巴
球釋放細胞激素之影響………………………………………………32
第四章討論…………………………………………………………………………. 34
結果之附圖……………………………………. ……………………………… 38
結果之附表……………………………………. ……………………………… 57
參考文獻……………………………………………………………………………. 67
附錄: 藥品配方……………………………………………………………………72

謝明村，中國藥材學，正中書局，1988；269-271。
時逸人，中國藥物學，台聯國風出版社，1972；442-443。
中藥藥理及應用，啟業書局，1985；554。
Baumann, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.J., Pulendran,
B.,and K., Palucka. (2000). Immunobiology of dendritic cells. Annu Rev Immunol.
18:767-811.
Baumann, J., Bruchhausen, F.V., and G., Wurm. (1980). Flavonoids and related
compounds as inhibitors of arachidonic acid peroxidation. Prostaglandins.
20:627-639.
Cao, L.Z., and Z.B., Lin. (2002). Regulation on maturation and function of dendritic
cells by Ganoderma lucidum polysaccharides. Immunol letter. 83:163-169.
Cho, S.Y., Park, S.J., Kwon, M.J., Jeong, T.S., Bok, S.H., Choi, W.Y., Jeong, W.I.,
Ryu, S.Y., Do, S.H., Lee, C.S., Song, J.C., and K.S., Jeong. (2003). Quercetin
suppresses proinflammatory cytokines production through MAP kinases and
NF-kappa B pathway in lipopolysaccharide-stimulated macrophage. Mol Cell
Biochem. 243:153-160.
Davis, W.L., and S.B., Matthew. (2000). Antioxidants and Cancer III:Quercetin.Altern
Med Rev. 5:196-208.
Dcnda, U.H., Tsujiuchi, T., Tsutsumi, M., Eimoto, H., Takashima, Y., Kitazawa, S.,
Kinugasa,T., and Y., Konishi. (1989). Possible involvement of arachidonic acid
metabolism in Phenobarbital promotion of hepatocarcinogenesis. Carcinogenesis.
10:1929-1935.
De Smedt, T., Pajak, B., Muraille, E., Lespagnard, L., Heinen, E., De Baetselier, P.,
Urbain, J., Leo, O., and M, Moser. (1996). Regulation of dendritic cell numbers and
maturation by lipopolysaccharide in vivo. J. Exp. Med. 184:1413-1424.
De Smedt, T., Pajak, B., Muraille, E., Lespagnard, L., Heinen, E., De Steven., D.,
Giuseppe, P., and L., Adorini. (2000). The control of T cell responses by dendritic cell
subsets. Curr Opin Immunol. 12:114-21.
Duperrier,K., Farre,A., Bienvenu, J., Bleyzac, N., Bernaud, J., Gebuhrer, L., Rigal, D.,
and A., Eljaafari. (2002). Cyclosporin A inhibits dendritic cell maturation promoted
by TNF-alpha or LPS but not by double-stranded RNA or CD40L. J Leukol Biol.
72:953-961.
Elangovan, V., Sekar, N., and S., Govindasamy. (1994). Chemopreventive potential of
dietary bioflavonoids against 20-methylcholanthrene-induced tumorigenesis. Cancer
Lett. 87:107-113.
Feili-Hariri,M.,Falkner, D.H., and P.A., Morel. (2002). Regulatory Th2 response
induced following adoptive transfer of dendritic cells in prediabetic NOD mice.Eur J
Immunol 32:2021-30.
Hanasaki, Y., Ogawa, S., and Fukui, S. (1994). The correlation between active
oxygens scavenging and antioxidative effects of flavonoids. Free Radic Biol Med.
16:845-850.
Hartgers, F. C., Figdor, C. G., Adema, G. J. (2000). Towards a molecular
understanding of dendritic cell immunobiology. Immunol Today. 21:542-5.
Hendriks, J.J., de Vries, H.E., van der Pol, S.M., van den Berg, T.K., van Tol, E.A.,
and C.D., Dijkstra. (2003). Flavonoids inhibit myelin phagocytosis by macrophages; a
structure-activity relationship study. Biochem Pharmacol. 65:877-885.
Hertog, M.G., Hollman, P.C., Katan, M.B., and D., Kromhout. (1993). Intake of
potentially anticarcinogenic flavonoids and their determinants in adults in the
Netherlands. Nutr. Cancer. 20:21-29.
Hollman, P.C., Hertog, M.G., and M.B., Katan. (1996). Role of dietary flavonoids in
protection against cancer and coronary heart disease. Biochem Soc Trans. 24:785-789.
Hou, Y.C., Chao, P.D.L., Ho, H.J., Wen, C.C., and S.L., Hsiu. (2003). Profound
difference in pharmacokinetics between morin and its isomer quercetin in rats. J
Pharm Pharmacol. 55:199-203.
Hsiu, S.L.,Tsao, C.W., Tsai, Y.C., Ho, H.J., and P.D.L., Chao. (2001). Determinations
of morin, quercetin and their conjugate metabolites in serum. Biol. Pharm. Bull. 24:
967-969.
Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S.,
and R.M., Steinman. (1992). Generation of large numbers of dendritic cells from
mouse bone marrow cultures supplemented with granulocyte /macrophage colony
stimulating factor. J. Exp. Med. 176:1693-1702.
Karen, A.O., Andrea, J.D., Paul, W.N., William, S.S., Nora, M.O., and W., Gary.
(2001). Flavonoid glucuronides are substrates for human liver β-glucuronidase.
FEBS letter. 503:103-106.
Kawabata, K., Tanaka, T., Honjo, S., Kakumoto, M., Hara, A., Makita, H., Tatematsu,
N., Ushida, J., Tsuda, H., and H., Mori. (1999). Chemopreventive effect of dietary
flavonoid morin on chemically induced rat tongue carcinogenesis. Int J Cancer.
83:381-386.
Kim, H.K., Cheon, B.S., Kim, Y.H., Kim, S.Y., and H.P., Kim. (1999). Effects of
naturally occurring flavonoids on nitric oxide production in the macrophage cell line
RAW 264.7 and their structure-activity relationships. Biochem Pharmacol. 58:759-
765.
Liang, L., and W.C., Sha. (2002). The right place at the right time: novel B7 family
members regulate effector T cell responses. Curr. Opin. Immunol. 14:384-390.
Ludovica., B., Thomas, S., and L., antinio. (2001). Mouse pre-immunocytes as
non-poliferating mutipotent precursors of macrophages,interferon-producing cells,
CD8α+and CD8α- dendritic cells. Eur. J. Immunol. 31:3403-3412.
Martha, T., and T., Kathy. (2002). Lipopolysaccharide recognition:CD14, TLRs and
the lps-activation cluster. Trends Immunol. 23:301-4.
Matsue, H., Yang, C., Matsue, K., Edelbaum, D., Mummert, M., and A., Takashima.
(2002). Contrasting impacts of immunosuppressive agents(rapamycin, FK506,
cyclosporin A, and dexamethasone) on bi-directional dendritic cell-T cell interation
during antigen presentation. J Immunol. 169:3555-3564.
Mu, M.M., Chakravortty, D., Sugiyama, T., Koide, N., Takahashi, K., Mori, I.,
Yoshida, T., and T., Yokochi. (2001). The inhibitory action of quercetin on
lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells.
J Endotoxin Res. 7:431-438.
Muriel, M., and K.M., Murphy. (2000). Dendritic cell regulation of TH1-TH2
development. Nat. Immunol. 1:199-205.
Nussenzweig, M.C., Steinman, R.M., Witmer, M.D., and B., Gutchinov. (1982). A
monoclonal antibody specific for mouse dendritic cells. Proc Natl Acad Sci U S A.
79:161-5.
Petersen, M.S., Toldbod, H.E., Hokland, M., Bolund, L., and R., Agger. (2000).
Strain-specific variations in the development of dendritic cells in murine bone-
marrow cultures. Scand. J. Immunol. 51:586-594.
Raso, G.M., Meli, R., Di Carlo, G., Pacilio, M., and R., Di Carlo. (2001). Inhibition of
inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in
macrophage J774A.1. Life Sci. 68:921-931.
Rissoan, M.C., Soumelis, V., Kadowaki, N., Grouard, G., Briere, F., de Waal Malefyt,
R., and Y.J., Liu. (1999). Reciprocal control of T helper cell and dendritic cell
differentiation. Science 283:1183-6
Robak, J., and R.J., Gryglewski. (1988). Flavonoids are scavengers of superoxide
anions. Biochem Pharmacol. 37:837-841.
Robinson, D., Allaway, S., Syms, J., and A.,Hale. (1995). Effect of red wine ingestion
on the antioxidant capacity of serum. Clin. Chem. 41:32-35.
Scambia, G., Ranelletti, F.O., Benedetti, P.P., Piantelli, M., Bonanno, G., De Vincenzo,
R., Ferrandina, G., Pierelli, L., Capelli, A., and S., Mancuso. (1991). Quercetin
inhibits the growth of a multidrug-resistant estrogen-receptor-negative MCF-7 human
breast-cancer cell line expressing type II estrogen-binding sites. Cancer Chemother
Pharmacol. 28:255-258.
Shen, S.C., Lee, W.R., Lin, H.Y., Huang, H.C., Ko, C.H., Yang, L.L., and Y.C., Chen.
(2002). In vitro and in vivo inhibitory activities of rutin, wogonin, and quercetin on
lipopolysaccharide-induced nitric oxide and prostaglandin E(2) production. Eur J
Pharmacol. 20:187-194.
Schipf, A., Heilmann, A., Boue, L., Mossmann, H., Brocker, T., and M., Rocken.
(2003). Th2 cells shape the differentiation of developing T cell responses during interactions with dendritic cells in vivo. Eur. J. Immunol. 33:1697-1706.
Sims, T.N., and M.L., Dustin. (2002). The immunological synapse: integrins take the
stage. Immunol Rev. 186:100-17.
Spencer, J.P., Chowrimootoo, G., Choudhury, R., Debnam, E.S., Srai, S.K., and C.,
Rice-Evans. (1999). The small intestine can both absorb and glucuronidate luminal
flavonoids. FEBS Lett. 458:224-230.
Stein, G.M., Bussing, A.,and M., Schietzel. (2002). Stimulation of maturation of
dendritic cells in vitro by a fermented mistletoe extract. Anticancer Res. 22:4215-
4219.
Steinman,R.M.,and Z.A., Cohn. (1973). Identification of a novel cell type in
peripheral lymphoid organs of ice:morphology,quantitation,tissue distribution. J. Exp.
Med. 137:1142-1162.
Steinman,R.M.,and Z.A.,Cohn. (1974). Identification of a novel cell type in peripheral
lymphoid organs of mice :functional properties in vitro. J. Exp. Med. 139:380-397.
Tanaka, T., Kawabata, K., Honjo, S., Kohno, H., Murakami, M., Shimada, R.,
Matsunaga, K., Yamada, Y., and M., Shimizu. (1999). Inhibition of azoxymethane-
induced aberrant crypt foci in rats by natural compounds, caffeine, quercetin and
morin. Oncol Rep. 6:1333-40.
Wang, H.K. (2000). The therapeutic potential of flavonoids. Exp. Opin. Invest. Drugs.
9:2103-2119.
Watson, D.G., and E.J., Oliveira. (1999). Solid-phase extraction and gas
chromatography- mass spectrometry determination of kaempferol and quercetin in
human urine after consumption of Ginkgo biloba tablet. J. Chromatogr. B, Biomed Sci.
Appl. 723:203-210.
Victor, D., and J., Winter. (1987). Hydrolysis of dietary flavonoid glycosides by
strains of intestinal bacteroides from human. Biochem. J. 248:953-956.