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研究生:文萬鈞
研究生(外文):Wen, Wanchun
論文名稱:表沒食子兒茶素沒食子酸脂透過抑制第六型介白素 減少 Porphyromonas gingivalis 之脂多醣體引起之牙齦纖維細胞產生第一型基質金屬蛋白酶
論文名稱(外文):Epigallocatechin-3-gallate Decreases the Porphyromonas gingivalis Lipopolysaccharide-induced Matrix Metalloproteinase-1 Production in Human Gingival Fibroblast Through the Inhibition of Interleukin-6
指導教授:傅鍔傅鍔引用關係
指導教授(外文):Fu, Earl
口試委員:張溫良江正陽涂筱培沈一慶聶鑫
口試委員(外文):Chang, WenliangChiang, ChengYangTu, HsiaopeiShen, EchinNieh, Shie
口試日期:2012-05-31
學位類別:碩士
校院名稱:國防醫學院
系所名稱:牙醫科學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:56
中文關鍵詞:表沒食子兒茶素沒食子酸脂第六型介白素脂多醣體牙齦纖維細胞第一型基質金金屬蛋白酶
外文關鍵詞:Epigallocatechin-3-gallatePorphyromonas gingivalis LipopolysaccharideMatrix Metalloproteinase-1Human Gingival FibroblastInterleukin-6
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綠茶主要是源自於Camellia sinensis此類植物,經由烘焙後製備成,其中的兒茶素類具有抗氧化的功效。在抗癌症、抗發炎方面已有文獻回顧。其中最有效的成分是表沒食子兒茶素沒食子酸酯Epigallocatechin-3-gallate(EGCG),占了兒茶素成分中的50%-75%。
牙周病主因由牙周致病菌引起宿主之一連串免疫反應而產生,首先造成結締組織之破壞進而齒槽骨吸收,而在牙齦組織中占最大多數比例的還是牙齦纖維細胞。經由文獻回顧牙周致病菌的LPS刺激牙齦纖維細胞後,會使細胞產生前驅發炎激素如IL-6,與一連串訊號傳遞下所衍生產物MMP-1,但是在牙齦纖維細胞的實驗模型上並未有完整的實驗探討此現象,因此本研究藉牙齦纖維母細胞以LPS刺激後產生出IL-6與MMP-1,進而使用EGCG做一抑制,以期達到抑制牙周炎的臨床效果,進而探討其路徑是否透過IL-6引起MMP-1增加,探討綠茶萃取物對於牙周病預防與治療的可能性。
實驗結果發現用牙周致病菌的LPS刺激使牙齦纖維細胞產生IL-6與MMP-1,進而利用外加入EGCG以降低IL-6與MMP-1的產量,不論是在蛋白質或是mRNA的幅度在加入EGCG 20μM時即有顯著的改變。而其可能的抑制機轉為IL-6生成受到影響而減低MMP-1合成。
BACKGROUND:
Green tea is produced from Camellia sinensis. And the most effective in reacting with reaction oxygen species and anti-inflammation is (-)-epigallocatechin-3-gallate, EGCG. Periodontal disease is an inflammatory disease may damage surrounding tissue and bone, causing tooth loss. In periodontitis, bacteria and their products, lipopolysaccharide, LPS can penetrate gingival connective tissue and induce a local inflammatory response that leads to periodontal bone resorption. Human gingival fibroblast are the most proportion in connective tissue, under lipopolysaccharide stimulation, HGFs will continuously releasing pro-inflammatory cytokines. Periodontitis is also in relation of host-immune systems. P. gingivalis LPS stimulated human gingival fibroblasts and found increased IL-6 and MMP-1 production. EGCG treatment showed anti-inflammation and anti-degradation in several studies. Decreased IL-6 synthesis and MMPs production were reported.
PURPOSE:
The aims of the study were 1) to evaluate the ameliorative effect of EGCG on the P.g LPS-enhanced MMP-1 production in human gingival fibroblasts, and 2) to further elucidate the role of IL-6 on ameliorative effect.
RESULTS:
Significantly increased MMP-1 amount was observed in HGFs after the stimulations of P.g LPS with a dose dependent manner. However, EGCG significantly reduced the LPS-enhanced MMP-1 amount. Significantly increased IL-6 mRNA expression was observed in HGFs after P.g LPS treatment. The IL-6 protein production was also increased after P.g LPS treatments. However, EGCG reduced the LPS-enhanced IL-6 mRNA and protein expressions. P.g LPS significantly increased MMP-1 protein production; however, the anti-IL-6 antibody inhibited the LPS-stimulated MMP-1. In addition, IL-6 treatment significantly enhanced the MMP-1 production, with a dose dependent manner.
CONCLUSION:
In the present study, EGCG inhibited the P.g LPS-induced MMP-1 production in HGFs through the regulation of IL-6. Thus may provide a possible prevention and treatment for periodontal disease through the anti-degradation ability.
正文目錄

第一章、
第一節、 表沒食子兒茶素兒茶酸酯------------------------------ 1
壹、 簡介 ------------------------------------------------- 1
貳、 體外細胞實驗之文獻回顧---------------------------------- 3
參、 動物模型實驗之文獻回顧---------------------------------- 5
肆、 臨床人體實驗之文獻回顧---------------------------------- 7
第二節、 金屬基質蛋白酶------------------------------------- 8
壹、 結構與功能--------------------------------------------- 8
貳、 與牙周病間之關係---------------------------------------- 9
第三節、 第六型介白素-------------------------------------- 10
壹、 結構與功能-------------------------------------------- 10
貳、 訊號傳遞機制------------------------------------------ 10
參、 與牙周病間之關係--------------------------------------- 11
第四節、 研究目的----------------------------------------- 11
第二章、 材料與方法---------------------------------------- 13
第三章、 實驗結果------------------------------------------ 23
第四章、 實驗討論------------------------------------------ 39
第五章、 結論--------------------------------------------- 45
第六章、 參考文獻----------------------------------------- 46

圖目錄
圖1、 EGCG對於人類牙齦纖維母細胞之毒性測試----------------------- 24
圖2、 EGCG抑制P. g LPS刺激牙齦纖維細胞使MMP-1之蛋白質生成量增加----- 26
圖3、 P. g LPS刺激牙齦纖維細胞使IL-6之mRNA之表現量變化------- 28
圖4、 P. g LPS刺激牙齦纖維細胞使第六型介白質之蛋白質生成量變化------------------ 30
圖5、 EGCG抑制P. g LPS刺激牙齦纖維細胞使IL-6之mRNA之表現量上升-------- 32
圖6、 EGCG抑制P. g LPS刺激牙齦纖維細胞使IL-6之蛋白質產生-------------- 34
圖7、 P. g LPS刺激牙齦纖維細胞,同時並加入抗IL-6抗體觀察MMP-1因LPS刺激而上升的現象,是否會因為抗IL-6抗體的加入而受到影響:--------- 36
圖8、 直接外加IL-6刺激牙齦纖維細胞使MMP-1之蛋白質生成量變化------------- 38
圖9、 加入不同濃度之EGCG於人類牙齦纖維細胞之型態變化---- 41

附錄目錄
附錄一、 表沒食子兒茶素兒茶酸酯(EGCG)的結構圖
附錄二、 EGCG可能的與細胞交互作用機制
附錄三、 綠茶兒茶素之各種異構物
附錄四、 上皮生長因子接受器可能的訊號傳遞路徑
附錄五、 IL-6引起訊號傳遞之示意圖
附錄六、 基質金屬蛋白酶的分類及其分泌細胞種類
附錄七、 人類牙齦纖維細胞受到LPS刺激經由細胞內訊號傳遞如p38與NF-κB進核,轉錄出IL-6 mRNA,並製造出IL-6細胞激素

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