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目的通过小鼠动物模型,探讨幽门螺杆菌(Helicobacter pylori,H.pylori)感染和用抗生素抗H.pylori治疗对食道下端菌群的影响,分析其与食道下端疾病发生的关系。方法将45只Balb/c小鼠随机均分为阴性对照组,感染组和治疗组。感染组和治疗组通过灌喂H.pylori建立动物感染模型,治疗组再灌喂奥美拉唑,氨苄青霉素,克拉霉素根除H.pylori。三组小鼠均在用抗生素处理后同时处死,取食道下端组织提取细菌的DNA,以原核生物16S rDNA V6区通用引物采用聚合酶链发应-变性梯度凝胶技术(PCR-DGGE)检测,用Quantity One1-DAnalysis software对DGGE图谱进行菌群结构分析,并将DGGE图谱上的组间差异条带用16S rDNA V6区引物分别扩增后,DNA测序,BLAST比对鉴定。结果成功制备了小鼠幽门螺杆菌感染模型,用抗生素有效根除了H.pylori感染。小鼠食道下端DGGE指纹图谱分析显示,各组间条带数量比较差异均有统计学意义(P<0.05),多样性指数、丰富度指数差异有统计学意义(0.05>P>0.001),均匀度指数差异无统计学意义(P>0.05)。菌群聚类分析能很好地在相似性进化树中分开,主成分分析不同组的菌群分别聚集在不同的位置,BLAST比对分析对照组,感染组具有特有细菌。结论食道下端定植着由大量细菌构成的较稳定的菌群,H.pylori感染与治疗后菌群结构有明显变化。
OBJECTIVE: To investigate the effect of Helicobacter pylori (H.pylori) infection and antibiotic-resistant H. pylori on the lower esophageal flora by animal model in mice and analyze the relationship with lower esophageal disease. Methods 45 Balb / c mice were randomly divided into negative control group, infection group and treatment group. Infected and untreated groups were infected with H.pylori to establish an animal model of infection. The treatment group was orally given omeprazole, ampicillin, and clarithromycin to eradicate H.pylori. All three groups of mice were sacrificed at the same time with antibiotic treatment. The bacterial DNA was extracted from the lower part of the esophagus. PCR-DGGE was used to detect the 16S rDNA V6 region universal primers of prokaryotes by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) The structure of DGGE was analyzed by Quantity One-DAnalysis software. The difference bands between two groups were amplified by 16S rDNA V6 primers, then sequenced and compared by BLAST. Results The mice model of Helicobacter pylori infection was successfully prepared and the antibiotics were effective in eradicating H.pylori infection. DGGE fingerprinting analysis of the lower esophagus in mice showed that there was significant difference in the number of bands between groups (P <0.05), and the difference of diversity index and richness index was statistically significant (0.05> P> 0.001) Degree index difference was not statistically significant (P> 0.05). Cluster analysis of bacteria can be well separated in the phylogenetic trees, principal components analysis of different groups of bacteria were clustered in different locations, BLAST comparison analysis of the control group, the infected group has endemic bacteria. Conclusions The lower esophagus is colonized by a relatively large number of bacteria, and the flora has obvious changes after H.pylori infection and treatment.