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研究生:陳佩旻
研究生(外文):Pei-Min Chen
論文名稱:環境壓力引發轉糖鏈球菌葡萄糖傳遞酶基因調控之分析
論文名稱(外文):Transcriptional Regulation of Streptococcus mutans Glucosyltransferase Genes in Response to Environmental Stress
指導教授:賈景山
指導教授(外文):Jean-San Chia
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:口腔生物科學研究所
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:2001
畢業學年度:89
語文別:中文
論文頁數:73
中文關鍵詞:轉糖鏈球菌葡萄糖傳遞酶
外文關鍵詞:Streptococcus mutansGlucosyltransferase
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轉糖鏈球菌是草綠色鏈球菌的一員,為造成人類齲齒的主要病原菌,經由代謝多種醣類而產生酸並能在酸性環境下生存,是其一個重要的毒性因子。轉糖鏈球菌也可能經由經常性的牙科手術或治療,而有機會進入血液中到達心臟,使已有不正常心臟瓣膜的病人引發感染性心內膜炎的發生。轉糖鏈球菌的葡萄糖傳遞酶會代謝蔗糖產生細胞外多糖體 ( 葡聚糖 ),此一具有黏性的物質會促使轉糖鏈球菌在口腔中的牙齒表面上形成緊密的附著,而當細菌進入血液中後,也能增加附著於心臟瓣膜上的贅生物,因此,轉糖鏈球菌葡萄糖傳遞酶基因的表現受環境壓力的調控,被認為對於引發毒性是很重要的研究主題。
本實驗利用北方墨點轉漬法 ( Northern blot ),從轉錄層次研究生長在對數期 ( exponential phase ) 的轉糖鏈球菌,其三個葡萄糖傳遞酶基因 ( gtfB,gtfC,gtfD ) 受不同壓力調控之表現。發現gtfB,gtfC,gtfD基因在轉錄層次的表現量不相同,而gtfC基因和gtfD基因在轉錄層次的調控不相同,面對壓力的改變外還受到培養液的影響,當細菌生長在酸性的M4培養液中,會使gtfC基因的表現量明顯增加,但是,當細菌生長在酸性的BHI培養液中,卻是gtfD基因的表現量明顯增加,另外,gtfC基因的表現量會受pH 7.4與pH 5.5的E培養液外加葡萄糖而減少,但和gtfD基因只在pH 5.5的E培養液外加葡萄糖才減少,且gtfC基因受影響較gtfD基因為大。此外,gtfD基因的表現量會受培養液外加citric acid,血漿,血清及銅離子而明顯增加。
雖然各種環境壓力對於轉糖鏈球菌的葡萄糖傳遞酶在轉錄層次的調控是相當複雜的,但本研究已初步找到重要調控的環境因子及其生長之條件。
Streptococcus mutans, a member of the viridans streptococci, is the primary aetiologic agent of dental caries. Acidogenesis through metabolism of multiple sugars and tolerance in the low pH are important virulent factors. S. mutans may enter the bloodstream during routine dental procedures and cause infective endocarditis in patients with heart valve abnormalities. The exopolysaccharide ( glucan ) formation through glucosyltransferases ( GTFs ) promotes tenacious adherence of S. mutans to the tooth surface in the oral cavity, and also enhances adherence to the vegetations present on the valves of the heart. Therefore, the regulation of GTF genes of S. mutans in response to environmental stress is hypothesized to be essential for virulence in vivo.
In this study, the transcriptional levels of the three genes ( gtfB, gtfC and gtfD ) in response to different stress challenges were analyzed by Northern blot in bacteria from exponential phase of growing. Differential expressions of gtfB, gtfC and gtfD at the transcriptional level were detected. Differential regulations of gtfC and gtfD at the transcriptional level were detected and were dependent on growth medium in addition to stress challenge. The gtfC but not gtfD gene was significantly up-regulated when S. mutans grown in defined media ( M4 media ) were exposed to acid stress. The gtfD gene was significantly up-regulated when S. mutans grown in rich media ( BHI media ) were exposed to acid stress. The expression of gtfC gene was subjected to catabolite repression by glucose in minimal media ( E media ) at pH 7.4 and pH 5.5. The expression of gtfD gene was subjected to catabolite repression by glucose only in minimal media ( E media ) at pH 5.5. The gtfD gene also was significantly up-regulated when S. mutans were treated with citric acid, serum, plasma, and in the presence of copper ion.
These results indicated that GTFs are subjected to complex regulation at transcriptional level in the presence of different nutritional factors and during stress response of S. mutans.
致謝……………………………………………………………….I
目錄……………………………………………………………...II
圖表目錄………………………………………………………..IV
中文摘要……………………………………………………….VI
英文摘要………………………………………….…………..VIII
壹、緒論…………………………………………………………..1
一、 轉糖鏈球菌之形態,特性,鑑定和分類……………………1
二、 轉糖鏈球菌與人類疾病的關係……………………………3
1. 齲齒…………………………………………………………3
2. 感染性心內膜炎……………………………………………5
三、 葡萄糖傳遞酶………………………………………………6
四、 葡萄糖傳遞酶與齲齒………………………………………8
五、 葡萄糖傳遞酶與感染性心內膜炎………………………..10
六、葡萄糖傳遞酶基因的調控…………………………………12
貳、實驗目的及設計……………………………………………20
參、實驗材料及方法……………………………………………21
一、 菌種與質體………………………………………………..21
二、 大腸桿菌 ( E.coli ) 質體 ( plamid ) 的抽取……………21
三、 純化DNA片段……………………………………………22
四、 放射性同位素標定探針 ( 32P-dCTP labeled probe )…...23
五、轉糖鏈球菌 ( Streptococcus mutans ) 核醣核酸 ( RNA ) 的抽取…….………………………………………………..24
六、核醣核酸 ( RNA ) 濃度偵測……………………………...26
七、北方墨點轉漬法 ( Northern blot )……………...………….26
八、轉糖鏈球菌 ( Streptococcus mutans ) 蛋白質 ( protein ) 的抽取…………………………………………………...……27
九、SDS-聚丙烯醯胺凝膠 ( SDS-PAGE ) 的製作…………....28
十、SDS-PAGE銀染色法 ( silver stain )…………….…………28
十一、西方墨點轉漬法 ( Western blot )……………………….29
肆、結果…………………………………………………………31
一、 轉錄層次 ( transcriptional level )………………………...31
1. 在不同培養液中酸刺激的影響…………………………..31
2. 不同濃度葡萄糖刺激的影響……………………………..31
3. 不同濃度葡萄糖及蔗糖濃度刺激的影響………………..33
4. 培養液中不同成分刺激的影響…………………..………33
5. 不同濃度血漿及血清刺激的影響………………………..34
6. 不同離子刺激的影響……………………………………..35
二、 轉譯層次 ( translational level )…………………………...35
1. 在BHI培養液中酸刺激的影響…………………………..35
伍、討論…………………………………………………………38
陸、參考文獻……………………………………………………45
圖表………………………………………………………..……54
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