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研究生:許凱杰
研究生(外文):Kai-Chieh Hsu
論文名稱:抗生素對於小鼠腸道上皮接合、黏液層和菌相的影響
論文名稱(外文):Effects of antibiotics on mouse gut epithelial junctions, mucus layer, and bacterial community
指導教授:何翰蓁
指導教授(外文):Han-Chen Ho
學位類別:碩士
校院名稱:慈濟大學
系所名稱:生理暨解剖醫學碩士班
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:60
中文關鍵詞:黏液層腸道菌穿透式電子顯微鏡
外文關鍵詞:mucus layergut microbiotatransmission electron microscopy
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小鼠大腸黏液層可分為內層黏液層及外層黏液層。內層黏液層貼附於上皮細胞表面,結構緻密且完全無細菌棲息;而外層黏液層較鬆散,有數量龐大的細菌分布其中。抗生素處理會改變宿主腸道菌相,臨床上亦常見抗生素治療引起腸發炎反應。然而,抗生素引起的腸道菌相改變是否也造成黏液層的變化,目前並不清楚。本實驗目的主要在探討宿主服用廣效性抗生素後,其腸道黏液層與腸道菌相之變化。老鼠連續七天餵食鏈黴素及桿菌肽後,生理狀態與控制組老鼠相似,並沒有發炎、腹瀉、體重減輕等現象,且腸道上皮間之緊密接合結構仍然完整,顯示腸道通透性仍維持正常。但在黏液層方面,抗生素處理組的老鼠,其前段及中段大腸的內層黏液層厚度與控制組相比有顯著的增加,而且外層黏液層中的細菌幾乎都已被消滅。我們也發現抗生素停藥七天後,三段大腸之內層黏液層厚度已回復至與控制組相同,同時外層黏液層中再次充滿細菌。在腸道菌相部分,各組別老鼠的前、中、後三段大腸菌相相似度高,在控制組老鼠大腸中主要以厚壁菌門 (Firmicutes) 的細菌為主,佔了約95%。其中超過50%為 Lachnospiraceae 及 Ruminococcaceae;約20% - 35%為Lactobacillaceae 及 Staphylococcaceae。抗生素處理後,三段大腸中細菌數量及種類均明顯變少,有超過90%為厚壁菌門之 Erysipelotrichaceae,其中又以 Turicibacter 屬之細菌為主要菌群,此現象或許可利用於發展研究 Turicibacter 之動物模式。至於停藥七天後,外層黏液層中除了細菌數量的回復外,腸道菌相亦由抗生素處理後之單純菌相再度回復到較複雜的菌相組成,雖然停藥七天後菌相與控制組仍有差異,但 Lachnospiraceae 及 Ruminococcaceae 等腸道菌已重新在老鼠腸道中聚生,形成主要菌群。
The intestinal mucus of mouse colon is organized into two layers. The inner layer is densely packed, firmly attached to the epithelium, and devoid of bacteria. In contrast, the outer layer is movable, and is colonized by bacteria. Antibiotic treatment is known to disturb gut microbiota and triggering inflammatory responses. However, whether changes of microbiota associated with mucus layer is hardly discussed. The aim of the study was to evaluate the impact of broad spectrum antibiotics on the gut epithelial junctions, mucus layer, and microbial community. Mice were treated with streptomycin and bacitracin (SB) for 7 days. Mouse colon was isolated and divided into proximal, middle, and distal parts. We found that SB-treated mice were physiologically normal: no diarrhea, body weight loss, tight junction dysfunction can be detected. SB-treated mice showed significantly increased inner mucus layer in proximal and middle colon, but not in distal colon. Besides, there was a dramatically decreased bacterial number in the outer mucus layers. Interestingly, the thickness of inner mucus layer retuned to control level, and the bacteria re-colonized at the outer mucus layer in SB-treated mice following antibiotic withdrawal for 7 days. To evaluate the SB on colon microbial community, bacterial DNA was extracted, 16S rDNA was PCR amplified, cloned and sequenced. We found that the major groups of bacteria are not differed in the 3 parts of colon: In control mice, Firmicutes dominate the community: over 50% are Lachnospiraceae and Ruminococcaceae; Lactobacillus and Staphylococcus made up 20-35% of the community. Surprisingly, SB effectively eliminated most of the gut bacteria, and the Turicibacter comprised over 90% of the survived bacteria. Furthermore, in SB-treated mice following antibiotic withdrawal, microbial community returned to a more complex structure similar to control mice. Although gut microbiota remained different from control, Lachnospiraceae and Ruminococcaceae quickly re-colonized the gut and became the dominant groups of bacteria.
中文摘要………………………………………………………………………I
英文摘要……………………………………………………………………II
1. 研究背景………………………………………………………………………………………………1
1.1 哺乳類動物之消化道………………………………………………………………………………1
1.2 胃腸道菌群的來源與分布…………………………………………………………………………2
1.3 腸道菌與宿主的關係………………………………………………………………………………2
1.4 腸道黏液層……………………………………………………………………………………………3
1.5 緊密接合………………………………………………………………………………………………4
1.6 抗生素………………………………………………………………………………………………5
1.7 抗生素對於腸道菌群的影響………………………………………………………………………6
1.8 抗生素對腸道黏液層的影響…………………………………………………………………………7
1.9 研究動機與目的……………………………………………………………………………………7
2. 實驗材料與方法……………………………………………………………………………………9
2.1 實驗動物………………………………………………………………………………………………9
2.2 實驗分組………………………………………………………………………………………………9
2.3 小鼠體重的監測與生理狀況的觀察………………………………………………………………9
2.4 樣本處理………………………………………………………………………………………………9
2.5 光學顯微鏡技術…………………………………………………………………………………10
2.6 穿透式電子顯微鏡技術…………………………………………………………………………11
2.7 內層黏液層厚度測量方法及統計分析……………………………………………………………12
2.8 腸道菌菌相分析……………………………………………………………………………………12
3. 結果……………………………………………………………………………………………………16
3.1 抗生素對老鼠腸道之影響…………………………………………………………………………16
3.2抗生素濃度對老鼠腸道的影響…………………………………………………………………17
3.3 停藥後黏液層的變化……………………………………………………………………………18
3.4停藥後腸道菌相之變化……………………………………………………………………………19
4. 討論……………………………………………………………………………………………………20
4.1 黏液層超微結構的保留…………………………………………………………………………20
4.2 黏液層在健康小鼠各腸段的差異…………………………………………………………………20
4.3 抗生素對老鼠腸道生理狀態之影響………………………………………………………………21
4.4 抗生素對腸道黏液層的影響………………………………………………………………………22
4.5 抗生素對腸道上皮細胞間緊密接合的影響……………………………………………………22
4.6 抗生素對腸道菌相的影響…………………………………………………………………………23
4.7停藥七天後對腸道黏液層之影響…………………………………………………………………23
4.8 停藥七天後對腸道菌相之影響……………………………………………………………………24
5. 結論…………………………………………………………………………………………………26
6. 參考文獻……………………………………………………………………………………………27
7. 附圖…………………………………………………………………………………………………32
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