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系統識別號 U0026-0812200915320107
論文名稱(中文) 幽門螺旋桿菌在上皮細胞中的複製及誘發細胞自噬
論文名稱(英文) The autophagic induction and multiplication of Helicobacter pylori in epithelial cells
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 97
學期 2
出版年 98
研究生(中文) 朱晏廷
研究生(英文) Yen-ting Chu
學號 S4696408
學位類別 碩士
語文別 中文
論文頁數 71頁
口試委員 口試委員-許秉寧
口試委員-吳俊忠
指導教授-黎煥耀
口試委員-林以行
中文關鍵字 胃上皮細胞  細胞自噬  抗生素  幽門螺旋桿菌 
英文關鍵字 antibiotics  Helicobacter pylori  autophagy  gastric epithelial cells 
學科別分類
中文摘要 全球有超過50% 人口受到幽門螺旋桿菌 (Helicobacter pylori) 感染,幽門螺旋桿菌感染中有部份會引起胃潰瘍或胃癌等疾病。過去研究曾經說明幽門螺旋桿菌會黏附於上皮細胞表面,甚至可以進入上皮細胞且存活於細胞質中的空泡,存在細胞內的幽門螺旋桿菌可以利用細胞內環境當作庇護所而躲避抗生素的破壞,進而引起長期的持續性感染,然而目前對於幽門螺旋桿菌是否會在上皮細胞中進行複製仍沒有直接的證據。在我們的實驗中顯示台灣臨床分離的HP238菌株可以在AGS以及MKN45的人類胃癌細胞株中進行複製,而且在感染後12小時有5倍的增加量,且在cagA及babA突變株也看到在上皮細胞中複製的現象,只是相對於HP238野生株則發現這兩種突變株進入細胞的量比較少,同時也會提早被細胞清除。另外我們也觀察到幽門螺旋桿菌感染後的AGS細胞質中會有雙層膜的構造圍繞著細菌,暗示幽門螺旋桿菌感染上皮細胞後會誘發細胞的自噬 (autophagy) 反應,並且觀察到細胞自噬體中有細菌的分裂,此外感染後的2.5小時就會有細胞自噬的特徵出現在幽門螺旋桿菌感染的細胞,例如LC3的聚集以及LC3-II的出現,有部份的幽門螺旋桿菌可以和溶小體 (lysosome) 後期的標示蛋白LAMP1在一起,以及p62蛋白質會隨時間減少,暗示細胞自噬體 (autophagosome) 會與溶小體融合,並分解胞內的幽門螺旋桿菌,以rapamicin處理可以抑制幽門螺旋桿菌在AGS細胞中的複製,因此我們推測幽門螺旋桿菌感染上皮細胞後會引起細胞自噬流體 (autophagic flux),而且細胞自噬體既是幽門螺旋桿菌的複製場所,也是細菌被分解的所在位置。觀察細胞膜上相連的幽門螺旋桿菌在感染後6到8小時之間有數目增加的現象,此外細胞膜附近也有細菌的分裂,而菌體轉變成球型體的數目會隨時間而增加,並由感染細胞的上清液中分離到細菌,因而推測細胞膜附近的幽門螺旋桿菌不但可以進行複製,還可以自由的離開細胞,並隨時間增加而轉變成球型體型態。此外我們也分別選用不同濃度的clarithromycin、metronidazole及 amoxicillin來評估臨床用的抗生素對於細胞內幽門螺旋桿菌的殺菌效果,並和傳統的最小抑制濃度 (MIC)及最小殺菌濃度 (MBC) 比較。使用五倍最小殺菌濃度的clarithromycin有明顯的抑制細胞內幽門螺旋桿菌的複製,而且隨著濃度增加效果越好。Metronidazole也有類似的細胞內抑制效果,但amoxicillin和金達酶素 (gentamicin) 則完全沒有抑制細胞內細菌複製的效果,可能和amoxicillin或金達酶素沒有能力擴散入細胞內有關。根據以上結果我們認為幽門螺旋桿菌進入細胞內複製,提供一個環境庇護抗生素的殺菌作用,細菌可能對於原本敏感性的抗生素濃度有了耐受性,因此要殺死細胞內的幽門螺旋桿菌需要更高濃度的抗生素,這新發現對於現今抗生素治療幽門螺旋桿菌的用法帶來新的看法。
英文摘要 Helicobacter pylori colonizes the stomach in about 50% of the world population and causes a variety of disease from gastric ulcers to cancer. It has been reported that H. pylori can adhere to the surface of gastric epithelium and be able to enter epithelial cells and survive within cytoplasmic vacuoles. The internalized H. pylori might utilize the intracellular environment to serve as a reservoir and cause long-term persistence despite antibiotic therapy. However, no multiplication of H. pylori is ever demonstrated. Here, we showed that the Taiwanese clinically isolated strain HP238 could multiply in human gastric cell line, such as AGS and MKN45, and there was a 5-fold increment at 12 h post infection after internalization. The cagA and babA mutant of H. pylori could also multiply within infected cells, but in a lower number and were quickly eliminated than the wild type. The double-membrane vesicles were observed within H. pylori-infected AGS cells and most H. pylori were found to reside in autophagosomes, suggesting that H. pylori might induce autophagy in epithelial cells. The characteristics of autophagic vesicle, LC3 aggregation or LC3-II conversion were observed as early as 2.5 h post infection. Some of LAMP1 positive vesicles containing H. pylori and decreased p62 were also observed within H. pylori-infected cells, suggesting autophagosome fused with lysosome. Rapamycin treatment could inhibit the multiplication of H. pylori in AGS cells. This suggests H. pylori infection induce the autophagic flux and autophagosome vesicles might supply a multiplicated niche as well as the digested sites for the intracellular H. pylori. The surface-associated H. pylori could be detected to increase at 6-8 h post infection and obtained from the infected AGS culture supernatant, implying that plasma membrane is one of niches that H. pylori can multiply and enter or exit it freely. Furthermore, the effects of clarithromycin, metronidazole or amoxicillin were examined on H. pylori-infected AGS cells. This intracellular inhibition of antibiotics was compared with the traditional minimal inhibitory concentration (MIC) or minimal bacteriocidal concentration (MBC). We found that clarithromycin could inhibit the multiplication of intracellular H. pylori at five times of MBC and has dose-dependent effect against intracellular H. pylori multiplication. Metronidazole also has some bactericidal effect for intracellular H. pylori. In contrast, amoxicillin or gentamicin do not have intracellular inhibitory effect due to its lack of membrane diffusion activity. This suggests that H. pylori residence in the cells might provide a resistance to antibiotic treatment. Only high concentrations of antibiotics with enough intracellular activity can help eliminating the intracellular H. pylori. Our new finding will have an impact on the current treatment of antibiotics to H. pylori infection.
論文目次 中文摘要.............................................I
英文摘要.............................................III
誌謝.................................................V
總目錄...............................................VI
圖目錄...............................................VIII
縮寫索引.............................................IX
緒 論................................................1
材料與方法...........................................16
一、細胞...........................................16
二、細菌...........................................16
三、幽門螺旋桿菌感染細胞...........................18
四、細胞內細菌的存活數目...........................18
五、流式細胞儀偵測細胞表面的細菌...................18
六、共軛焦螢光顯微鏡的觀察.........................19
七、西方墨點法.....................................21
八、穿透式電子顯微鏡的樣本製備.....................22
九、抗生素敏感性試驗 - 最低抑菌濃度測定............22
十、抗生素對細胞內細菌存活的影響...................23
十一、儀器.........................................24
十二、藥品與試劑...................................25
結 果................................................28
一、幽門螺旋桿菌可以在上皮細胞進行複製.............28
二、毒力因子影響幽門螺旋桿菌在上皮細胞中的複製以及存活...................................................28
三、細胞膜附近的幽門螺旋桿菌可以自由進出細胞.......29
四、幽門螺旋桿菌位於上皮細胞內的雙層膜構造中.......30
五、幽門螺旋桿菌的感染誘發上皮細胞的細胞自噬 (autophagy) 反應.................................................31
六、上皮細胞中的幽門螺旋桿菌對抗生素殺菌能力的影響.32
討 論................................................34
一、幽門螺旋桿菌可被視為一種細胞內微生物.............34
二、細胞內幽門螺旋桿菌的存活.........................35
1. 毒力因子 (virulence factor).....................35
2. 膽固醇 (cholesterol)............................36
3. 球形體幽門螺旋桿菌..............................37
三、幽門螺旋桿菌在上皮細胞引起的細胞自噬.............38
1. 細胞自噬在幽門螺旋桿菌感染中的角色..............38
2. 啟動細胞自噬的可能原因..........................39
四、存在上皮細胞內的幽門螺旋桿菌對抗生素殺菌能力的影響..41
五、結 論............................................42
參考文獻.............................................43
圖...................................................57
作者簡歷.............................................71
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