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研究生:顏加豪
研究生(外文):Jia-Hau
論文名稱:金門一條根對細胞激素及清除凋亡細胞之效果
論文名稱(外文):Effects of Glycine tomentella Hayata extract on cytokines and clearance of apoptotic cells
指導教授:蔡嘉哲蔡嘉哲引用關係
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:免疫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:51
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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

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