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研究生(外文):Yi-Ying Tsai
論文名稱(外文):Studies of signaling molecules between TLR and MAPK in HepG2 cells infected with Klebsiella pneumoniae
指導教授(外文):June-Hsieh Wu
外文關鍵詞:Klebsiella pneumoniaeMAPKToll-like receptorsHepG2TLRsliver abscessp44/42p38
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在台灣,克雷白氏肺炎桿菌 (klebsiella pneunomina, KP) 是化膿性肝膿瘍 (pyogenic liver abscess) 的主要致病菌,國外少見此種現象。實驗室的興趣在於釐清KP與肝細胞的關係和KP在肝細胞中引起的訊息傳遞現象。之前實驗室利用HepG2作為感染系統,發現活菌KP可引起細胞當中的p44/42、p38傳遞路徑之活化和TLR2/TLR4高度的表現;在此實驗中我們由KP分離的莢膜多醣體 (capsule polysaccharide) 單獨存在發現可活化p44/42、p38,但只刺激TLR4表現。在抗體抑制性試驗 (感染後30分鐘) 的結果當中,發現抑制TLR2及TLR4後,HepG2細胞之p44/42 MAPK及p38 kinase路徑之活化可被少量的抑制,但以純化之KP LPS來與細胞作用時,TLR4抗體可明顯的顯示p44/42及p38路徑被抑制。猜想在活菌作用時,莢膜多醣體的立體結構可能部份擋住位於克雷白氏肺炎桿菌上TLR2和TLR4的配體 (ligand),另外或許也可能有其他受體在全菌感染時參與下游p44/42和p38的磷酸化,致使反應不明顯。在免疫沉澱試驗中,以KP LPS與HepG2作用(感染後30分中),可觀察到MyD88及TRAF6相互作用,但未能觀察到MyD88與TAK1的交互作用,此部分需進一步的研究。
Severe liver abscess caused by Klebsiella pneumoniae (KP) has been observed recently in Taiwan. How the bacteria cause liver abscess is not clear. Our laboratory previously used human hepatocellular carcinoma cell line, HepG2, as a model system to study the signal transduction during infection and found that infecting HepG2 cell with whole KP bacteria caused upregulation of TLR2 and TLR4 protein expression and activation of p38 kinase and p44/42 MAPK pathways. In this experiment, we found that bacterial capsule polysaccharide purified from KP can activated p44/42, p38 and TLR4 but not TLR2. We did not observe the downstream molecules, MyD88, IRAK1, TRAF6, TAK1 interacting with TLR2 or TLR4 in HepG2 cells infected with live whole bacteria by immunoprecipitation experiment. However, in HepG2 cells treated with KP LPS, the interaction of Myd88 and TRAF6 could be observed. Inhibition assay with TLR2 or TLR4 antibodies indicated that both p38 kinase and p44/42 MAPK activations were inhibited slightly at antibody concentration of 20μg/ml, however, such inhibition can be observed clearly when the whole bacteria infection was substituted with LPS form KP. It seems that capsule polysaccharide may form steric hinderance to prevent bacterial LPS from activation downstream molecules. The association of other molecules, such as IRAK1, TAK1, MyD88 and TRAF6 with TLR2 or TLR4 needs further investigation.
緒論 1
ㄧ、克雷白氏肺炎桿菌的介紹 1
二、台灣地區的克雷白是肺炎桿菌特有的肝膿 2
三、克雷白氏肺炎桿菌的致病因子 3
四、由細菌感染引發的訊息傳遞 8
甲、Toll-like receptors 8
TLR4 10
TLR2 10
TLRs所引發的訊息傳遞 11
乙、MAPK kinase 14
丙、NF-κB 16
研究目的 21
實驗材料與方法 22
實驗結果 27
討論 54
結論 61
參考文獻 62
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