臺灣博碩士論文加值系統

牙周病是由於細菌的侵襲，導致宿主免疫系統反應。牙周組織發炎反應持續，會導致牙齦萎縮、牙周附著喪失與齒槽骨流失，最終導致牙齒脫落。
牙齦卟啉單胞菌為牙周病致病菌之一，為革蘭氏陰性菌。咖啡目前被發現具有抑制細菌生長及減緩發炎反應的能力。
本研究針對不同的兩株牙齦卟啉單胞菌進行實驗，檢測咖啡對於牙齦卟啉單胞菌生長能力的影響。以抑菌圈實驗檢測牙齦卟晽單胞菌對於咖啡的敏感程度，將兩株牙齦卟啉單胞菌塗抹於培養皿上，把沾取不同濃度咖啡的濾紙片貼附其上，厭氧培養24小時後測量直徑大小；之後以抑菌實驗檢測咖啡對於牙齦卟啉單胞菌生長的影響，將兩株牙齦卟啉單胞菌與不同濃度的咖啡混合後，塗抹於培養皿上，厭氧培養24小時後計算細菌顆數。本研究更針對細胞產生的發炎反應進行實驗，檢測咖啡對於受脂多醣刺激的細胞產生的發炎反應的影響。以免疫試驗檢測咖啡對於細胞產生的發炎因子的影響，實驗的發炎因子有四項:TNF-a、IL-1a、IL-6、IL-8，將細胞組分為四組培養24小時:對照組、加咖啡組、加脂多醣組、加咖啡與脂多醣組，取上清液進行免疫測試，最後以酵素免疫分析儀以OD450nm讀測其數值。
實驗結果顯示兩株牙齦卟啉單胞菌對於咖啡的敏感性皆顯著高於對照組，且咖啡相較於對照組及純水組皆有顯著抑制兩株牙齦卟啉單胞菌的生長。而在免疫測試得出咖啡可以顯著抑制細胞產生TNF-a的量，並且可以顯著抑制受刺激的細胞產生IL-6及IL-8。本研究顯示咖啡可以抑制牙齦卟啉單胞菌的生長，且對於細菌引起的發炎因子有抑制的作用

Coffee is a popular drink currently shown its abilities of anti-bacterium and anti-inflammation. Periodontal disease is a result of the host immune response to bacterial invasion. If periodontal tissue inflammation continues, the disease will lead to gingiva recession, periodontal attachment loss and alveolar bone destruction, eventually leading to the exfoliation of the tooth. Porphyromonas gingivalis, a Gram-negative bacterium, is one of the bacteria associated with periodontal disease.
To test the ability of coffee to inhibit the growth of P. gingivalis in this study, we measured the sizes of the bacterial growth inhibition zone to evaluate the sensitivity of two strains of P. gingivalis to different concentrations of coffee by the method of filter disc diffusion. We also co-cultivated P. gingivalis with coffee on the anaerobic agar plates and counted the colony forming unit of bacteria after 24 hours incubation. ELISA experiments were conducted to quantify four kinds of inflammatory cytokines, TNF-a, IL-1a, IL-6, and IL-8, produced by LPS-stimulated osteoblast-like cells in the presence or absence of coffee to evaluate the effect of coffee on the cell inflammatory responses.
Our results showed that P. gingivalis was significantly sensitive to coffee and coffee inhibited the growth of P. gingivalis. Coffee significantly inhibited the production of TNF-a and inhibited the LPS-stimulated cells to produce IL-6 and IL-8.
In conclusion, coffee can inhibit the growth of periodontitis associated bacterium P. gingivalis even in a very diluted concentration and reduce the production of pro-inflammatory cytokines and alleviate the inflammatory responses of already activated cells.

目錄
中文摘要 ii
Abstract iii
致謝.. iii
目錄 iv
表目錄 vi
圖目錄 vii
第一章 前言 1
1.1 牙周病背景介紹 1
1.2 牙周致病菌及毒力因子-Lipopolysaccharide 3
1.3 牙周病的病因..4
1.4 發炎物質: TNF-a、IL-1a、IL-6、IL-8.. 5
1.5 咖啡的主要成分.. 6
1.6 咖啡與牙周病的關係.. 7
1.7 實驗目的.. 8
第二章 實驗材料與方法
2.1 實驗材料與藥品製備.. 9
2.1.1 Wilikins Chalgren anaerobe.. 9
2.1.2 Wilikins Chalgren agar plate.. 9
2.1.3 Human TNF-a、IL-1a、IL-6、IL-8 ELISA development kit plate... 9
2.2 實驗樣本分析.. 10
2.2.1 P.g.菌的培養..10
2.2.2 咖啡的製備..10
2.2.3 抑菌圈實驗.. 10
2.2.4 抹盤實驗.. 11
2.2.5 LPS的製備.. 11
2.2.6 細胞的培養..11
2.2.7 Human TNF-a、IL-1a、IL-6、IL-8 ELISA test.. 11
2.2.8 統計分析.. 12
第三章 實驗結果.. 13
3.1 P.g.菌33277抑菌圈實驗.. 13
3.2 P.g.菌A7436抑菌圈實驗.. 13
3.3 P.g.菌33277抑菌實驗.. 14
3.4 P.g.菌A7436抑菌實驗.. 15
3.5 ELISA test : TNF-a.. 16
3.5.1 ELISA test : TNF-a(DMEM/DMEM + coffee)..16
3.5.2 ELISA test : TNF-a(DMEM/DMEM + LPS)..16
3.5.3 ELISA test : TNF-a(DMEM + LPS/DMEM + LPS + coffee).. 16
3.6 ELISA test : IL-1a..17
3.6.1 ELISA test : IL-1a(DMEM/DMEM + coffee).. 17
3.6.2 ELISA test : IL-1a(DMEM/DMEM + LPS)..17
3.6.3 ELISA test : IL-1a(DMEM + LPS/DMEM + LPS + coffee).. 17
3.7 ELISA test : IL-6.. 18
3.7.1 ELISA test : IL-6(DMEM/DMEM + coffee).. 18
3.7.2 ELISA test : IL-6(DMEM/DMEM + LPS).. 18
3.7.3 ELISA test : IL-6(DMEM + LPS/DMEM + LPS + coffee).. 18
3.8 ELISA test : IL-8..19
3.8.1 ELISA test : IL-8(DMEM/DMEM + coffee).. 19
3.8.2 ELISA test : IL-8(DMEM/DMEM + LPS).. 19
3.8.3 ELISA test : IL-8(DMEM + LPS/DMEM + LPS + coffee).. 19
第四章 討論.. 21
4.1 抑菌圈實驗.. 21
4.2 抑菌生長實驗..22
4.3 ELISA test.. 24
4.4 ELISA test : TNF-a.. 24
4.5 ELISA test : IL-1a.. 25
4.6 ELISA test : IL-6.. 26
4.7 ELISA test : IL-8.. 27
第五章 結論 29
第六章 參考文獻 30
第七章 附錄..33
表目錄
各實驗成分表..34
表一 Wilikins Chalgren anaerobe broth 成分表 34
表二 Wilikins Chalgren agar成分表 34
表三 Dulbecco’s Modified Eagle Medium製備表 35
表四 Human TNF-a、IL-1a、IL-6、IL-8 ELISA development kit溶液製備表35
表五 Human TNF-a、IL-1a、IL-6、IL-8 ELISA development kit材料表35
表六 Human TNF-a ELISA development kit藥品成分製備表35
表七 Human IL-1a ELISA development kit藥品成分製備表36
表八 Human IL-6 ELISA development kit藥品成分製備表37
表九 Human IL-8 ELISA development kit藥品成分製備表37 
圖目錄
實驗結果圖..39
圖1 測試P.g.菌菌株33277對於不同濃度咖啡的敏感性 39
圖2 測試P.g.菌菌株A7436對於不同濃度咖啡的敏感性 39
圖3 測試不同濃度咖啡對於P.g.菌菌株33277生長的影響40
圖4 測試不同濃度咖啡對於P.g.菌菌株A7436生長的影響40
圖5 測試咖啡對於正常細胞產生TNF-a的影響 41
圖6 測試正常細胞受LPS刺激後產生TNF-a的影響 41
圖7 測試咖啡對於正常細胞受LPS刺激後產生TNF-a的影響 42
圖8 測試咖啡對於正常細胞產生IL-1a的影響 42
圖9 測試正常細胞受LPS刺激後產生IL-1a的影響 43
圖10 測試咖啡對於正常細胞受LPS刺激後產生IL-1a的影響 43
圖11 測試咖啡對於正常細胞產生IL-6的影響 44
圖12 測試正常細胞受LPS刺激後產生IL-6的影響 44
圖13 測試咖啡對於正常細胞受LPS刺激後產生IL-6的影響 45
圖14 測試咖啡對於正常細胞產生IL-8的影響 45
圖15 測試正常細胞受LPS刺激後產生IL-8的影響 46
圖16 測試咖啡對於正常細胞受LPS刺激後產生IL-8的影響 46

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