营养性肥胖大鼠模型肠道菌群的微生物多样性分析

Analysis of intestinal microbiota diversity in nutritional obesity rats

目的肠道菌群与营养性肥胖关系密切,但多数研究集中在大肠粪菌,小肠作为吸收的主要场所,其菌群还缺乏研究,本文通过微生物多样性分析,探索回肠与结肠菌群差异及营养性肥胖相关菌群。方法混合喂养法建立营养性肥胖SD大鼠模型,chow继续喂养60 d消除HFD的影响,提取回肠、结肠内容物总DNA,PCR扩增16S rRNA基因V3+V4区并测序,建立OTUs,通过Silva数据库进行注释和分类学分析。结果(1)alpha分析显示回肠菌群数量高于结肠(chao1、ace),而多样性低于结肠(simpson、shannon)(P<0.05);(2)beta分析显示回肠与结肠以及回肠样本间物种相似度较低;(3)OTUs注释及聚类分析显示回肠与结肠优势菌(丰度排名top 10)的类型和丰度分布重叠性较低(属水平);(4)HFD-OR大鼠回肠Rothia丰度增加,而Romboutsia丰度降低。结论回肠与结肠具有不同的菌群多样性;Rothia菌和Romboutsia菌可能是参与肥胖发生的关键回肠菌群。

Objective The intestinal flora is closely related to nutritional obesity.Most of the previous research has focused on the fecal flora of the large intestine,with very few studies on the flora of the small intestine,which is the main site of absorption.We aimed to investigate the differences in bacterial flora between the colon and the ileum in a rat model of nutritional obesity using microbial diversity analysis.Methods Male Sprague-Dawley(SD)rats were fed with a high-fat diet(HFD)mixed with chow for 60 days to establish a nutritional obesity model,then fed with chow for another 60 days to eliminate the influence of the HFD.Total DNA was extracted from the contents of the ileum and colon and used as template for amplification of the V3+V4 region of the 16 S rRNA gene by PCR,then sequenced to establish operational taxonomic units(OTUs).Annotation and taxonomic analysis were performed against the Silva database.Results(1)Alpha analysis showed that the number of intestinal flora was higher than that of colon(chao1,abundance-based coverage estimator),but the diversity was lower than that of the colon(simpson’s Index,shannon function)(P<0.05);(2)Beta analysis showed that the species similarity between the ileum and colon+ileum samples was low;(3)OTU annotation and cluster analysis showed that there were obvious differences in the types and species abundance distributions of dominant bacteria(top 10 in abundance)in the ileum and colon,with low overlap at the genus level;(4)The abundance of Rothia in the ileum of HFD-obesity resistant rats increased significantly,while that of Romboutsia decreased.Conclusions The ileum and colon had different levels of diversity in intestinal flora in rats with nutritional obesity.Rothia and Romboutsia may be the key ileum flora involved in obesity.