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研究生:劉鴻毅
研究生(外文):Hong-yi Liu
論文名稱:蒜頭伯克氏菌體與培養液中3-OH脂肪酸之定量與角色之研究
論文名稱(外文):Study on the roles and quantification of 3-hydroxy fatty acids in the soma and supernatant of cultured Burkholderia cepacia complex
指導教授:陳亞雷
指導教授(外文):Ya-Lei Chen
學位類別:碩士
校院名稱:國立高雄師範大學
系所名稱:生物科學研究所
學門:生命科學學門
學類:生物學類
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:65
中文關鍵詞:劉鴻毅伯克氏菌群脂多醣體氣相層析質譜C14:0 3-OH 脂肪酸
外文關鍵詞:Hong-yi LiuBurkholderia cepacia complexlipopolysaccharideGas chromatography mass spectrometryC14:0 3-OH fatty acid
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蒜頭伯克氏菌體與培養液中3-OH脂肪酸之定量與角色之研究

摘要

伯克氏菌群(Burkholderia cepacia complex, BCC)現今使用recA-genomovar分類為9群,但genomovar的分類並無法區別BCC菌群的毒性。本研究假設BCC培養液之C14:0 3-OH脂肪酸(C14:0 3-OH FA;BCC脂多醣體的獨特成分)濃度,可作為生物指標物,資以判定菌株感染宿主後,誘發發炎之病理程度。因此,選擇經生化與分子確認檢測為gonomorvarⅢa之BCC菌株,並以脂肪酸組成與RAPD (randomly amplified polymorphic DNA)圖譜分析,確認32菌株具有基因獨立的代表性。茲為準確的定量培養液FAs濃度,本研究發展以氣相層析質譜儀定量脂肪酸脂化物(FAME)的方法,最低C14:0 3-OH FAME檢測濃度為26 ng/ml。最佳分析條件是取菌株培養7d後之濾清液的濃縮物,經鹼水解30min與脂化10min的條件下,可得到大於90%的酯化率。BCC菌群依此檢測條件定量培養液之C14:0 3-OH FA的濃度為19.3±0.4-133.7±3.6ng/ml;C14:0/C16:0 3-OH FA莫耳比為1.78±0.3。並進一步以多變數回歸分析顯示,C14:0 3-OH FA的濃度與BCC感染Balb/c小鼠2d後,所造成之肝臟發炎的病理切片特徵(發炎細胞碎片面積)呈正相關(R2=0.68)。本研究結果顯示菌株培養液之C14:0 3-OH FA濃度,可作為判定BCC感染Balb/c小鼠2d後的毒性指標。
關鍵字:伯克氏菌群、脂多醣體、C14:0 3-OH fatty acid、氣相層析質譜
Study on the roles and quantification of 3-hydroxy fatty acids in the soma and supernatant of cultured Burkholderia cepacia complex

Abstract

Although Burkholderia cepacia complex (BCC) divided into 9 of recA genomovars in present, the virulence to hosts in these genomovars were still undifferentiated. We hypothesized that, in cultural supernatants, the amounts of C14:0 3-OH fatty acids, the unique components of BCC lipopolysaccharides (LPS), acted as a biomarker for virulence, were related to the degrees of pathologically inflammatory effects on animals with BCC infection. Thirty-two strains were identified to BCC gonomorvarⅢa by the outcomes of biochemical and molecular tests, and typed to their genetic independence by the profiles of cellular fatty acid and randomly amplified polymorphic DNA (RAPD), respectively. To determine the concentration of fatty acids in supernatants, the quantitative profiles of each fatty acid methyl ester (FAME) analyzed by gas chromatography mass spectrometry (GC/MS) was developed. In this study, the instrument detection limits were reached to 26 ng/ml. The optimal reaction was defined as >90% in esterification for each fatty acid. The processing of samples were performed as that, after a 7 d-incubation, the cultural supernatants were acted as reactants and sequentially reacted to alkaline hydrolysis for 30 min and esterification for 10min. Among of the cultures of these isolates, the concentration of C14:0 3-OH fatty acid in supernatants was ranged from 19.3±0.4 to 133.7±3.6ng/ml. The molar ratio of C14:0/C16:0 3-OH fatty acid was calculated to be 1.78±0.3. With multiple regression analysis, a positive correlation was shown that the concentration of 3-OH fatty acids in supernatants of each isolates was against some levels of pathological effect (area of cell debris in liver) of Balb/c mice with tested BCC infection individually (R2=0.682). Results suggested that the concentration of C14:0 3-OH fatty acid in BCC cultural supernatants was acted as an indicator of virulence to the mice with the bacterial infection for 2 d.
Keywords:Burkholderia cepacia complex, lipopolysaccharides, C14:0 3-OH fatty acid, Gas chromatography mass spectrometry
目錄
致謝------------------------i
中文摘要--------------------ii
英文摘要--------------------iii
目錄----------------------- iv
圖目錄-----------------------v
表目錄-----------------------vi
第一章、 前言-----------------1
第二章、 研究目的--------------6
第三章、 材料與方法------------7
第四章、 結果-----------------14
第五章、 討論-----------------20
第六章、 參考文獻--------------26
圖---------------------------33
表---------------------------47
附錄-------------------------55
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