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研究生:潘靚穎
研究生(外文):Jing-Ying Pan
論文名稱:甘松精油對於突變型鏈球菌致病能力之影響
論文名稱(外文):Effect of Spikenard essential oils on virulence of Streptococcus mutans
指導教授:蔡英傑蔡英傑引用關係
指導教授(外文):Ying-Chieh Tsai
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
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:79
中文關鍵詞:突變型鏈球菌甘松致病能力
外文關鍵詞:Streptococcus mutansspiekenardvirulence
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牙菌斑是由牙齒表面堆積的生物膜所構成,會引起齲齒的發生。突變型鏈球菌是主要形成齲齒的重要病源之一,具有多種與齲齒發生相關的致病能力,包括:黏附作用、形成生物膜、產酸作用及耐酸反應。
利用體外篩選系統,篩選數十種實驗室現有的精油,得到幾種能夠抑制突變型鏈球菌黏附作用與形成生物膜的精油。其中抑菌效果顯著的有甘松、雪松及檜木精油等,最後選擇甘松精油作為抑制突變型鏈球實驗的材料。利用矽膠管柱層析的方法將全精油成份分成幾個不同的部分再進行黏附於多孔盤的生物質量測試,顯示Fraction 2具有最佳的抑制效果。往後的實驗選擇甘松全精油及Fraction 2作更進一步抑制突變型鏈球菌生成生物膜的活性測試。另外利用氣相層析質譜儀分析甘松全精油及Fraction 2的內含物,發現倍半萜類佔最大宗。
甘松全精油及Fraction 2的最小抑菌濃度(MIC)分別為139μg/ml與273.4μg/ml。實驗結果指出無論是甘松全精油或是Fraction 2,隨精油濃度的增加,抑制突變型鏈球菌的生物質量、菌體存活率與非水溶性葡聚醣產量有顯著的正相關。
突變型鏈球菌的葡萄糖基轉移酶和果糖基轉移酶主要參與胞外多醣的形成。利用反轉錄PCR分析這些基因的表現量,顯示在甘松全精油及Fraction 2較高濃度之sub-MIC中,gtf-B 和 gtf-C基因的表現量有些許受到精油抑制的現象。然而,ftf 基因的表現量只受到Fraction 2高濃度的sub-MIC所抑制。在甘松精油的作用下,突變型鏈球菌生成生物膜之致病能力有明顯減弱的情形,說明了甘松精油有潛力作為預防齲齒發生的用途。
Dental plaque is a complex biofilm that accumulates on the hard tissues (teeth) in the oral cavity and leads to dental caries. Streptococcus mutans is regarded as the principal etiologic agent of human dental caries. There are many major virulence properties of S. mutans associated with caries formation process include adhesion, biofilm formation, acidogenicity, and acid tolerance.
Dozens of essential oils (E.O.s) were screened. Several candidates of E.O.s such of spikenard, sandalwood and sedarwood E.O.s had effects on reducing attached S. mutans biomass in in vitro microtiter-plate screening test. Spikenard E.O. was chosen to be a target material to test inhibition of virulence of S.mutans. The spikenard E.O. was fractionated by silica column chromatography and fractions were tested the effect of reduction attached biomass. Fraction 2 was the most effective portion of all fractions. Spikenard E.O. and Fraction 2 later were be tested the activity of inhibition of S. mutans’s virulence properties. The major compounds of spikenard E.O. and Fraction 2 are sesquiterpene determined by using gas chromatography-mass spectrometry..
Minimum inhibitory concentration (MIC) of spikenard E.O. and Fraction 2 were 139�慊/ml and 273.4�慊/ml respectively. Spikenard E.O. and Fraction 2 could substantially reduce attached biomass, total viability and WIG content in a dose-dependent manner under sub-MIC.
The glucosyltransferase and fructosyltrasferase enzymes of S. mutans catalyze the formation of extracellular polymers. RT-PCR analysis for the expression of these genes showed expression of gtf-B and gtf-C genes can be inhibited under high sub-MIC of spikenard E.O. and Fraction 2. However ftf gene expression was attenuated only by Fraction 2 under high sub-MIC. The significant attenuation of S. mutans’s biofilm formation by spikenard E.O. demonstrated that spikenard E.O. would be a potent natural product for the prevention of dental caries.
中文摘要................................................ 1
英文摘要................................................ 3
一、 研究背景......................................... 5
1. 齲齒與突變型鏈球菌................................ 5
2. 突變型鏈球菌致病能力特性.......................... 6
2.1. 黏附.......................................... 6
2.2. 共黏附........................................ 8
2.3. 生物膜........................................ 8
2.4. 產酸能力..................................... 11
2.5. 耐酸反應..................................... 12
2.6. 其他......................................... 12
3. 植物萃取物與抗菌作用.............................. 13
4. 植物精油、抗菌作用與口腔保健...................... 18
5. 研究動機與目的.................................... 20
6. 實驗設計.......................................... 21
二、 實驗方法與材料................................... 23
1. 實驗材料.......................................... 23
1.1. 菌株與培養方式............................... 23
1.2. 實驗藥品..................................... 24
2. 儀器設備......................................... 25
3. 實驗方法......................................... 25
3.1. 培養基與藥品配製............................. 25
3.1.1. BHI培養基............................... 25
3.1.2. 結晶紫染劑(crystal violet)................... 26
3.1.3. XTT 試劑................................ 26
3.2. 有效全精油與有效精油分離部分之測定........... 26
3.3. 管柱層析法................................... 28
3.4. 氣相層析質譜儀分析........................... 29
3.5. 最低抑菌濃度測定............................. 31
3.6. 黏附之活菌量測量(XTT assay)................... 31
3.7. 生物膜中非水溶性葡聚醣(WIG)含量測定.......... 32
3.8. 萃取RNA..................................... 34
3.9. 反轉錄PCR................................... 35
三、 實驗結果......................................... 35
1. 能有效降低黏附之生物質量的全精油與精油分離部分的篩選................................................ 36
2. 甘松全精油與Fraction 2的最低抑菌濃度.............. 36
3. 甘松全精油和Fraction 2對黏附之生物質量的影響......37
4. 甘松全精油和Fraction 2對黏附菌體之總活菌量的影響..38
5. 甘松全精油和Fraction 2對於生物膜中非水溶性葡聚醣含量的影響........................................... 38
6. 甘松全精油與Fraction 2對突變型鏈球菌之致病能力基因表現量的影響....................................... 39
四、 討論............................................ 40
1. 牙菌斑、齲齒與口腔傷害........................... 40
2. 有效精油與精油分離部分的篩選..................... 41
3. 甘松全精油與Fraction 2的最小抑菌濃度與低於最小抑菌濃度............................................... 43
4. 甘松全精油與Fraction 2對黏附生物質量、總活菌量及胞外多醣含量之影響................................... 44
5. 甘松全精油與Fraction 2對減少黏附的生物質量及胞外多醣的機制推測....................................... 46
6. 甘松全精油與 Fraction 2抑制突變型鏈球菌生物膜形成之機制確認與實用價值................................. 49
五、 參考文獻........................................ 50
六、 附表與附圖...................................... 55
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