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研究生:蕭雅文
研究生(外文):Hsiao, Ya-Wen
論文名稱:肺炎克雷白氏桿菌莢膜、纖毛、脂多醣交互作用探討
論文名稱(外文):Investigation Of The Interaction Among Capsule, Fimbriae, And Lipopolysaccharide In Klebsiella pneumoniae
指導教授:蕭樑基蕭樑基引用關係林永崇林永崇引用關係
指導教授(外文):Siu, Leung-KeiLin, Jung-Chung
口試委員:蕭樑基林永崇林靖婷陳盈璁陳政男
口試委員(外文):Siu, Leung-KeiLin, Jung-ChungLin, Ching-TingChen, Ying-TsongChen, Cheng-Nan
口試日期:2012-06-28
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
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:91
中文關鍵詞:肺炎克雷白氏桿菌莢膜第一型纖毛第三型纖毛O抗原黏附侵入
外文關鍵詞:Klebsiella pneumonaecapsuletype 1 fimbriaetype 3 fimbriaeO antigenadhesioninvasion
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  • 下載下載:8
  • 收藏至我的研究室書目清單書目收藏:1
肺炎克雷白氏桿菌是常見的伺機性感染菌,引起的疾病包括敗血症、肺炎、以及尿道炎。在過去二十年間,侵襲性症候群,一種被定義為肺炎克雷白氏桿菌菌血症合併肝膿瘍、腦膜炎、以及眼內炎的疾病,在台灣感染比例有攀升的趨勢。
目前已證實K1與K2血清型與肺炎克雷白氏桿菌引起的侵入性症候群有高度的相關性。然而,有研究顯示,在引起尿道感染的臨床菌株中,K1/K2血清型比例較低。儘管肺炎克雷白氏桿菌的毒力因子已相當明確,包括莢膜、黏附蛋白、脂多醣,但這些構造與尿道炎的關係仍未明朗。對於泌尿道致病性大腸桿菌引起尿道感染的致病過程而言,黏附在泌尿道上皮細胞是重要的步驟。在本研究中,探討莢膜、第一型纖毛、第三型纖毛與O抗原是否與肺炎克雷白氏桿菌黏附能力有關。
首先,我們以一株K1血清型的肺炎克雷白氏桿菌,1001S作為原始菌株,製造了莢膜、第一型纖毛、第三型纖毛與O抗原突變株。即時定量聚合酶連鎖反應用於確認基因剔除。分別應用葡萄糖胺酸定量、酵母菌凝集、生物膜形成以及銀染試驗,證實突變株喪失了莢膜、第一型纖毛、第三型纖毛與O抗原。黏附試驗與侵入試驗中,證實莢膜突變株貼附與侵入腎臟上皮細胞能力較高。然而,喪失第一型纖毛、第三型纖毛與O抗原的突變株,黏附與侵入能力沒有顯著下降。研究結果證實莢膜遮蔽效應影響第一型莢膜血清型肺炎克雷白氏桿菌黏附與侵入的能力。


Klebsiella pneumoniae is an opportunistic pathogen which can cause septicemia, pneumonia, and urinary tract infections (UTIs). Over the past two decades, the invasive syndrome, which is characterized by community -acquired K. pneumoniae bacteremia with liver abscess, metastatic meningitis, and endophthalmitis, has emerged in Taiwan.
It has been demonstrated that the serotype K1 and K2 is strongly associated with K. pneumoniae related invasive syndrome. However, some studies showed a low prevalence of the serotypes K1/K2 in UTIs isolates. Although a number of virulence factors have been identified in K. pneumoniae, including capsule, adhesin, and lipopolysaccharide (LPS), it is not clear how these structures are associated with UTIs. Adherence to uroepithelial cell of uropathogenic Escherichia coli (UPEC) has been described to important for the establishment of UTIs. In this study whether capsule, type 1 fimbriae, type 3 fimbriae and O anigen were correlated with K. pneumoniae adherence was investigated.
Initially, we constructed several mutants including capsule (ΔK), type 1 fimbriae (ΔF1) , type 3 fimbriae (ΔF3) and O anigen (ΔO) from a serotype K1, encapsulated K. pneumoniae strain 1001S. Real-time quantitative polymerase chain reaction (Q-PCR) was used to confirm the genes were deleted. Glucuronic acid quantification, yeast agglutination, biofilm formation, and silver stain were used to assess the mutants that had loss their capsule, type 1 fimbriae, type 3 fimbriae and/or O antigen. Both adhesion and invasion assay showed that capsule mutants displayed a higher ability to attach and invade kidney epithelial cell. However, the mutants loss their type 1 fimbriae, type 3 fimbriae, and O antigen did not show significant decrease in adhesion and invasion. We conclude that capsule shielding effect was highly correlated with adhesion and invasion ability of serotype K1 K. pneumoniae.

第一章 緒論 1
第一節 肺炎克雷白氏桿菌的特性 2
第二節 莢膜多醣 3
第三節 脂多醣 4
第四節 攜鐵蛋白 5
第五節 纖毛 6
第六節 尿道炎 8
第七節 本研究的假設及目標 9
第二章 材料與方法 11
第一部份 材料 12
第一節 菌株 12
第二節 質體 12
第三節 引子 12
第四節 細胞株 12
第五節 試藥 13
第六節 儀器 16
第七節 試劑配方 17
第八節 培養基配方 21
第二部份 方法 23
第一節 聚合酶連鎖反應 23
第二節 製備突變用質體 24
第三節 熱休克轉型作用 25
第四節 製備突變株 26
第五節 即時定量聚合酶連鎖反應 27
第六節 生長速率的測定 28
第七節 葡萄糖醛酸萃取與定量試驗 29
第八節 酵母菌凝集實驗 31
第九節 生物膜合成試驗 32
第十節 細菌脂多醣體萃取 33
第十一節 SDS-PAGE電泳 33
第十二節 銀染 34
第十三節 細胞培養 34
第十四節 黏附試驗 34
第十五節 侵入試驗 35
第十六節 統計分析 36
第三章 結果 38
第一節 製造突變菌株 39
第二節 基因剔除後的基因表現量確認 40
第三節 菌株的莢膜多醣體生成量比較 41
第四節 菌株的第一型纖毛活性比較 42
第五節 菌株的第三型纖毛活性比較 43
第六節 菌株的脂多醣體生成量比較 44
第七節 突變菌株黏附與侵入細胞能力的比較 44
第四章 討論 47
第一節 肺炎克雷白氏桿菌黏附與侵入細胞的能力 48
第二節 肺炎克雷白氏桿菌表面結構的相互調節 52
第三節 肺炎克雷白氏桿菌表面結構的毒力影響 53
第五章 結論及未來研究 55
圖表 57
參考文獻 78
附錄 91

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