目的:
一條根學名又稱“Glycine tomentella Hayata”(GTH)，為台灣及金門傳統治療風濕病之草藥，廣泛應用在民間，本研究目的是探討一條根對抗風濕及發炎之機轉。
實驗方法:
我們使用95%乙醇萃取一條根，用HPLC對一條根萃取物做分析。在細胞實驗方面，使用老鼠巨噬細胞株 RAW264.7及人類單核球細胞株U937當做實驗對象，將一條根萃取物以乙醇回溶後加入細胞，先以MTT assay 觀察細胞存活率，再以RT-PCR分析脂多醣(lipopolysaccharide； LPS)引起發炎的模式下，對細胞激素IL-1β, IL-6 , TNF-α，第二型轉麩醯胺酶(Transglutaminase type2；TG2)和誘導性一氧化氮合成酶(iNOS) 的mRNA表現；ELISA分析IL-1β和IL-6 蛋白質分泌層級的變化。以Gelatin- zymography分析基質金屬蛋白酶(matrix metalloproteinase-2；MMP-2)和MMP-9之活性。最後以RAW264.7 做吞噬能力分析，以螢光乳膠粒子(latex-beads)和凋亡細胞(apoptotic cells)作為吞噬對象，了解一條根萃取物萃取物對 RAW264.7對螢光乳膠粒子和凋亡細胞之吞噬能力之影響。
實驗結果:
我們的實驗結果發現一條根萃取物在濃度330μg /ml時會對RAW264.7細胞造成存活率造成下降(p﹤0.05)。在LPS引起發炎的模式下以RT-PCR檢測，一條根萃取物在濃度165μg /ml對IL-6有56%之抑制、對IL-1β有55%之抑制，但對iNOS及TNF-α則否，另外，第二型轉麩醯胺酶在一條根萃取物對在LPS刺激下對 RAW264.7有47%抑制的現象，但在無LPS刺激下一條根萃取物對第二型轉麩醯胺酶增強58%。在人類的單核球細胞株U937，一條根萃取物對IL-6 有79%之抑制。對基質金屬蛋白酶， 在RAW264.7中MMP-9活性有受一條根萃取物57%之抑制，但對MMP-2並無明顯影響。吞噬作用上，對凋亡細胞的吞噬能力增加156%，但對於螢光乳膠粒子則否。
結論:
一條根萃取物在LPS刺激下，有減少發炎細胞激素IL-1β、IL-6，降低MMP-9的活性，增加apoptotic cells之清除能力，這些結果證實一條根萃取物有抗風濕及發炎之功能，並可推廣應用於風濕疾病之治療。

Objective :
I-Tiao-Gung , Glycine tomentella Hayata(GTH) , has been used as a traditional herbal medicine to treat rheumatic diseases in Kinmen and Taiwan, indicative of its anti-inflammatory effect. However , the possible mechanism of the pharmacological effect has not been completely delineated. The aim of this research is to investigate the effects of GTH on proinflammatory cytokines production in mouse macrophage cell line RAW264.7 and human monocyte cell line U937.

Methods :
Mouse macrophage cell line RAW264.7 and human monocyte cell line U937 were cultured with lipopolysaccharide(LPS) in the presence or absence
of ethanol extract of I-Tiao-Gung. The gene expression of proinflammatory cytokines: IL-1β, IL-6,TNF-α ,iNOS and transglutaminase 2 (TG2) were assayed by reverse transcriptase-polymerase chain reaction. The production of IL-1β, IL-6 ,TNF-α and metalloproteinase-9 (MMP-9) were assayed by ELISA and gelatin zymography.The phagocytosis activiti of RAW264.7 was analyzed by ingesting apoptotic cells and latex-beads and then detected at 4h by FACS.

Results :
Our results showed that the ethanol extract of GTH 165μg/ml inhibited the gene expression as well as the production of proinflammatory cytokines: IL-1β(55%), IL-6(56%) in LPS-activated macrophages, but only IL-6 (79%)in LPS-activated human monocyte. Furthermore, the gene expression of TNF-α and iNOS had no significant difference compared with the LPS-activated cell. In addition, the ethanol extract of GTH also inhibited the production of proinflammatory mediators, transglutaminase 2 (TG2)(47%) and metalloproteinase-9 (MMP-9) (57%) in LPS-activated macrophages. Morever, TG2 mRNA expression was enhanced 58% than control in the treatment wth GTH alone.In the experiment of phagocytosis, it was found that RAW264.7 had higher ability to ingest apoptotic cells (156% enhanced) and no significant difference in latex-beads.

Conclusion :
In our study ,we found the ethanol extract of GTH suppressed the LPS-induced production of IL-1β, IL-6 , transglutaminase 2 (TG2) and metalloproteinase-9 (MMP-9)in mouse macrophage cell line RAW264.7. The inhibition of IL-6 also exerted in human monocyte cell line U937. The clearance of apoptotic cells was also enhanced. This would indicate the anti-inflammatory potencies of this traditional herbal remedy.

目錄
壹、中文摘要................................ ................................ ......................... 1
貳、英文摘要································ ································ ························· 3
參、緒論································ ································ ································ 5
一、一條根(Glycine tomentella Hayata) ···························· 5
二、關節炎之成因與細胞激素································ ···················· 5
三、細胞凋亡與吞噬作用、抗原清除在慢性發炎自體免疫疾病中
所扮演之角色································ ································ ······ 6
四、基質金屬蛋白酶、組織型轉麩胺脢tissue Transglutaminase (tTG)
與發炎反應································ ································ ··············· 7
肆、研究動機································ ································ ························ 10
伍、實驗材料及方法
一、材料
1. 材料及藥品································ ································ ·11
2. 細胞株來源································ ································ ·12
二、方法
陸、實驗結果
一、一條根對細胞存活率之影響································ ·················19
二、一條根對發炎前細胞激素之影響································ ·········21
三、一條根對基質金屬蛋白酶-9 之影響································ ·····21
四、一條根對吞噬作用之影響································ ·····················22
柒、討論································ ································ ·······························24
捌、圖表
表一、以HPLC高效液相層析儀分析一條根萃取物。·····················31
圖一、一條根萃取物對RAW264.7細胞株細胞生長能力的影響········32
圖二、一條根萃取物處理RAW264.7細胞株24小時之後，加入LPS刺
激細胞，其IL-1β、IL-6、TG2 mRNA的表現量。················33
圖三、觀察一條根萃取物處理U937細胞株24小時之後，加入LPS刺
激細胞，其IL-1β、IL-6、TG2 mRNA的表現量。················35
圖四、Fig3.觀察一條根萃取物處理RAW264.7細胞株24小時之後，
加入LPS刺激細胞，其IL-6 protein的表現量。·············36
圖五、觀察一條根萃取物處理RAW264.7細胞株24小時之後，加入LPS
刺激細胞，其MMP-9及MMP-2的活性。································ ··37
圖六、利用螢光顯微鏡觀察老鼠巨噬細胞株(RAW264.7) 吞噬
Latex-beads 及凋亡細胞的情形及吞噬能力·····························38
圖七、觀察一條根萃取物處理RAW264.7 細胞株24 小時之後，加入
latex-beads 或凋亡細胞 4 小時後其phagocytosis 的功能。·39
玖、參考文獻

1..Lu, T. C., Y. Z. Ko, H. W. Huang, Y. C. Hung, Y. C. Lin, and W. H. Peng. 2007. Analgesic and anti-inflammatory activities of aqueous extract from Glycine tomentella root in mice. In J Ethnopharmacol. 142-148.

2.Ko, Y. J., Y. W. Wu, and W. C. Lin. 2004. Hypolipidemic effect of Glycine tomentella root extract in hamsters. The American journal of Chinese medicine 32:57-63.

3.Sun Pan, B., Y. Y. Kuo, T. Y. Chen, and Y. C. Liu. 2005. Anti-oxidative and anti-inflammatory activities of two different species of a Chinese herb I-Tiao-Gung. Life sciences 77:2830-2839.

4.Chen, T. Y., M. S. Shiao, and B. S. Pan. 2005. Inhibition of 12- and 15-lipoxygenase activities and protection of human and tilapia low density lipoprotein oxidation by I-Tiao-Gung (14). Lipids 40:1171-1177.

5.Chung, E. Y., S. J. Kim, and X. J. Ma. 2006. Regulation of cytokine production during phagocytosis of apoptotic cells. Cell research 16:154-161.

6.Emlen, W., J. Niebur, and R. Kadera. 1994. Accelerated in vitro apoptosis of lymphocytes from patients with systemic lupus erythematosus. J Immunol 152:3685-3692.

7. Chia, W. T., Y. W. Chen, et al. (2008). "MMP-9 mRNA as a therapeutic marker in
acute andchronic stages of arthritis induced by type II collagen antibody." J
Formos Med Assoc 107(3): 245-52.

8.Kerr, J. R., V. S. Cunniffe, P. Kelleher, A. J. Coats, and D. L. Mattey. 2004. Circulating cytokines and chemokines in acute symptomatic parvovirus B19 infection: negative association between levels of pro-inflammatory cytokines and development of B19-associated arthritis. Journal of medical virology 74:147-155.

9.Lorenz, H. M., M. Grunke, T. Hieronymus, M. Herrmann, A. Kuhnel, B. Manger, and J. R. Kalden. 1997. In vitro apoptosis and expression of apoptosis-related molecules in lymphocytes from patients with systemic lupus erythematosus and other autoimmune diseases. Arthritis and rheumatism 40:306-317.

10.Nunes, I., P. E. Gleizes, C. N. Metz, and D. B. Rifkin. 1997. Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta. The Journal of cell biology 136:1151-1163.

11.Nunes, I., R. L. Shapiro, and D. B. Rifkin. 1995. Characterization of latent TGF-beta activation by murine peritoneal macrophages. J Immunol 155:1450-1459.

12.Perniok, A., F. Wedekind, M. Herrmann, C. Specker, and M. Schneider. 1998. High levels of circulating early apoptic peripheral blood mononuclear cells in systemic lupus erythematosus. Lupus 7:113-118.

13.Szondy, Z., Z. Sarang, P. Molnar, T. Nemeth, M. Piacentini, P. G. Mastroberardino, L. Falasca, D. Aeschlimann, J. Kovacs, I. Kiss, E. Szegezdi, G. Lakos, E. Rajnavolgyi, P. J. Birckbichler, G. Melino, and L. Fesus. 2003. Transglutaminase 2-/- mice reveal a phagocytosis-associated crosstalk between macrophages and apoptotic cells. Proceedings of the National Academy of Sciences of the United States of America 100:7812-7817.

14.Tauber, A. I. 2003. Metchnikoff and the phagocytosis theory. Nature reviews 4:897-901.

15.Zou, J. J., R. J. Singh, and T. Hymowitz. 2004. SSR marker and ITS cleaved amplified polymorphic sequence analysis of soybean x Glycine tomentella intersubgeneric derived lines. TAG. Theoretical and applied genetics 109:769-774.

16.Fadok, V. A., D. L. Bratton, A. Konowal, P. W. Freed, J. Y. Westcott, and P. M. Henson. 1998. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. The Journal of clinical investigation 101:890-898.

17. Rose, D. M. Sydlaske, A. D. et al, Transglutaminase 2 limits murine peritoneal acute gout-like inflammation by regulating macrophage clearance of apoptotic neutrophils, Arthritis Rheum, 2006. 54 (10): p 3363-71.

18. Akimov, S.S. and A.M. Belkin, Cell surface tissue transglutaminase is involved in adhesion and migration of monocytic cells on fibronectin. Blood, 2001. 98(5): p. 1567-76.

19.Hsu, T. C., W. J. Wu, M. C. Chen, and G. J. Tsay. 2004. Human parvovirus B19 non-structural protein (NS1) induces apoptosis through mitochondria cell death pathway in COS-7 cells. Scand J Infect Dis 36:570
.
20. Tóth B, Garabuczi E, Sarang Z, Transglutaminase 2 is needed for the formation
of an efficient phagocyte portal in macrophages engulfing apoptotic cells.. 2009
Feb 15;182(4):2084-92.

21. Ram M, Sherer Y, Shoenfeld Y. J Clin Immunol. Matrix metalloproteinase-9 and
autoimmune diseases. Epub 2006 May 2. Review. 2006 Jul;26(4):299-307.

22 Yu X, Lin SG, Huang XR, Bacher M, Leng L, Bucala R, Lan HY. Macrophage
migration inhibitory factor induces MMP-9 expression in macrophages via the
MEK-ERK MAP kinase pathway. J Interferon Cytokine Res. 2007
Feb;27(2):103-9.

23 Žiaková, A., Brandsteterová, E., Blahová, E. 2003. Matrix solid-phase
dispersion for the liquid chromatographic determination of phenolic acids in
Melissa officinalis. J. Chromatogr. A 983: 271-275

24 Chuang, W. L., O. Haugland, B. S. Pan, and O. Evensen. 2008. Isoflavone-rich extracts from wooly glycine Glycine tomentella inhibits LPS-induced TNF-alpha expression in a macrophage cell line of Atlantic salmon (Salmo salar L.). Molecular immunology 45:3956-3964.