臺灣博碩士論文加值系統

克雷伯氏肺炎桿菌為革蘭氏陰性菌，在醫院為常見引起院內感染的重要病原菌。其易引起化膿性感染、肺炎、泌尿系統及敗血症，即使投予抗生素之治療後，仍有相當高的死亡率。然而，近年來在台灣克雷伯氏肺炎桿菌肝膿瘍之發生率卻有逐年增加趨勢，並且釵h克雷伯氏肺炎桿菌肝膿瘍患者皆發生肝外轉移病症，而造成很嚴重的合併症，如敗血性內眼炎導致失明。比較國內外發現 如此大的差異與高死亡率，頗受國內感染專家學者的重視。而在近年的文獻報告中，指出三個與肝膿瘍發生有關的致病因子，分別是rmpA、rmpA2、與magA。因此，我們利用跳躍子(mini-Tn5)與克雷伯氏肺炎桿菌進行random mutagenesis獲得一株跳躍子突變株。藉由血清抗性試驗結果得知此突變株對人類血清有高感受性，並由0.3% soft agar 培養基可發現突變株的菌落直徑明顯比野生株大。值得注意的是，將野生株與突變株以腹腔注射方式打入小鼠體內，結果顯示突變株毒性明顯有下降情形。並利用ELISA去測定小鼠肺部組織及血清內的細胞激素，結果指出野生株與突變株在動物模式中細胞激素表現量有很明顯的差異，藉由以上實驗結果間接說明此突變株與調節宿主免疫防禦機制有關並且推測其是一個與肺炎致病力有關的突變株，因此，將此突變株命名為pvf (pneumonia virulence factor) 突變株。

Klebsiella pneumoniae is an important pathogen of nosocomial and community-acquired infections, especially suppurative infection, pneumonia, urinary tract infection, and septicemia in humans. Althogh the development of new and potent antibiotics, K. pneumoniae infectious are still associated with a high mortality rate up to 50%. In Taiwan, the high incidence of K. pneumoniae pyogenic liver abscess has never reported in English literature. In addition, extrahepatic metastases, such as septic endophthalmitis, are often occurred with serious complications. The high mortality rate and high incidence of K. pneumoniae pyogenic liver abscess in Taiwan make further investigation is necessary. The current researches, we have known several virulence factors of K. pneumoniae liver abscess : (1) rmpA; (2) rmpA2; (3) magA. In this study, the pvf (pneumoniae virulence factor) mutant was isolated by mini-Tn5 transposon insertional inactivation and a serum sensitivity assay. The pvf mutant was significantly more easily killed by human serum than the wild type. The colony diameter of this mutant on 0.3% soft agar plate was larger than those of wild type strain. Interestingly, the mice experiments demonstrated virulence attenuation when the pvf mutant was administered intraperitoneally to normal mice. In addition, the cytokine expression profile in lung tissue of mice infected by pvf mutant showed significantly different from those of mice infected by wild type, suggesting that the pvf factor may mediate resistance to host innate defenses and it is an important virulence factor for mice.

中文摘要 I
英文摘要 II
誌謝 IV
目錄 VI
圖表目錄 IX
縮寫表 X
第一章 緒論 1
（一）克雷伯氏肺炎桿菌(Klebsiella pneumoniae) 1
（二）克雷伯氏肺炎桿菌致病機轉 2
（三）血清殺菌系統(serum bactericidal system) 3
（四）細胞激素 (cytokine) 4
（五）研究動機 6
第二章 材料與方法 7
（一）使用之菌株、載體及培養基 7
（二）染色體DNA之抽取 9
（三）染色體DNA以Sau3AI作部分切割（partial digest） 10
（四）製備小量質體 11
（五）DNA濃度測定 12
（六）聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 13
（七）限制酶切割質體DNA 14
（八）接合反應 (Ligation) 16
（九）轉形作用 (Transformation) 17
（十）基因突變株之構築與篩選 18
（十一）移動性實驗 (Motility Assay) 19
（十二）血清耐受性試驗 (Serum resistance test) 20
（十三）老鼠致死性之測定 21
（十四）支氣管肺泡灌洗樣本(BAL)及血清之收集（Bronchoalveolar lavage and serum collection） 23
第三章 結果 25
（一）構築mini-Tn5突變株 25
（二）比較克雷伯氏肺炎桿菌野生株及突變株表現型及致病力 26
表現型 26
游動性試驗 26
比較野生株及突變株菌落黏性 26
致病力 27
血液耐受性試驗 27
老鼠半致死率測定 (LD50) 27
（三）動物模式 28
（四）菌量測定 28
（五）細胞激素表現量測定 29
（六）組織切片 30
第四章 討論 31
第五章 結論 33
參考文獻 34
圖表 42
附錄 55

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