瑞士乳杆菌调控小鼠肠道菌群变化规律的研究

Modulation of Probiotic Lactobacillus helveticus on Gut Microbiota in Mice

为探究瑞士乳杆菌(Lactobacillus helveticus)TS206对小鼠肠道菌群的调控规律,更好地阐述益生菌维持肠道菌群稳态的机制,本研究以雄性BALB/c小鼠为实验对象,将其分为L.helveticus TS206组、对照组、空白组。L.helveticus TS206组和对照组分别每天灌胃L.helveticus TS206菌悬液和等量的生理盐水,连续灌胃7周,空白组不灌胃。每周采集小鼠新鲜粪便样品,通过试剂盒提取粪便细菌总DNA,利用Ion torrent个人化操作基因组测序平台测序技术对16S r RNA基因的V6区进行高通量测序,最后通过生物信息学和多变量统计学方法对测序数据进行分析。结果显示:所有测序序列在97%相似水平划分得到1 617个操作分类单位(operational taxonomic units,OTUs),被划分为8个门,硬壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)为各组小鼠中的优势菌门,占总序列数的97.49%,且硬壁菌门丰度最高,超过70%;优势菌科主要为S24-7、毛螺菌科(Lachnospiraceae)、瘤胃球菌科(Ruminococcaceae)、理研菌科(Rikenellaceae)和拟杆菌科(Bacteroidaceae),且与对照组相比L.helveticus TS206组肠杆菌科细菌数量较低并呈下降趋势。经LEf Se分析有53个关键OTUs与2组小鼠肠道粪便菌群结构显著相关,其中27个OTUs在对照组中富集,分属于肠杆菌科、瘤胃球菌科、毛螺菌科和梭菌目;26个OTUs在L.helveticus TS206组中富集,分属于梭菌目、毛螺菌科和拟杆菌属,其中与对照组相比,梭菌目丰度水平高。经主成分分析,两组样品完全分离,菌群的整体结构存在差异,且富集的OTUs之间呈负相关关系。综上所述,在实验阶段,灌胃雄性BALB/c小鼠一定剂量的L.helveticus TS206后,经扩增子测序分析获得的初步结论为L.helveticus TS206能改变肠道菌群整体结构的功效,表现出了抑制肠道有害微生物生长的作用,并通过促进部分有益菌的增殖来维持肠道菌群结构的稳态。

Objective： To explore the role of the probiotic strain Lactobacillus helveticus TS206 in regulating the intestinal microfiora in mice and to unravel the mechanisms by which probiotics can maintain intestinal fiora homeostasis. Methods： Male BALB/c mice were divided into probiotic, control and blank groups. The probiotic and control groups were respectively gavaged with L. helveticus suspension and an equal volume of normal saline once a day for 7 consecutive weeks, respectively. The blank group did not receive gavage. Stool samples were collected every week for extracting genomic DNA by means of a commercial kit. The V6 variable region of the 16S rRNA gene was subjected to high-throughput sequencing by using the Ion torrent personal genome machine system and the obtained data were analyzed using bioinformatic tools and multivariable statistical analysis. Results： All sequence reads were delineated into 1 617 operational taxonomic units （OTUs） at a 97% similarity level, belonging to 8 major bacterial phyla, with Firmicutes and Bacteroidetes being the dominant ones in all groups, accounting for 97.49% of the total number of sequences, amongwhich Firmicutes showed the highest abundance of more than 70%. The strain S24-7, Lachnospiraceae, Ruminococcaceae, Rikenellaceae, and Bacteroidaceae were the dominant bacterial families, and the Enterobacteriaceae population in the probiotic group was lower than in the control group and fell during the administration period. Totally 53 key OTUs were igni？cantly associated with the bacterial community structure in the intestine of the two groups of mice as determined by inear discriminant analysis effect size （LEfSe） analysis, of which, 27 OTUs were enriched in the control group, belonging o Enterobacteriaceae, Ruminococcaceae, Lachnospiraceae and Clostridium, and 26 OTUs in the L. helveticus TS206 group, belonging to Clostridium, whose abundance level was higher than in the control group, Lachnospiraceae and Bacteroides. Principal component analysis showed that the fecal microbial communities of the probiotic group were completely separated rom those of the control group. Overall there were signi？cant differences in the structure of intestinal ？ora between the two groups, and the OTUs in the groups showed a negative correlation. Conclusion： L. helveticus TS206 can change the structure of intestinal ？ora, inhibiting the growth of harmful microbes, promoting the proliferation of beneficial bacteria proliferation nd consequently maintaining the structure of the intestinal ？ora at a steady state.