基于16SrDNA扩增子测序探究脱氧胆酸对肠型胃癌模型INS⁃GAS小鼠胃内菌群结构的影响
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国家自然科学基金(81970499,81770561);江苏省医学领军人才与创新团队(CXTDA2017033);江苏省六大人才高峰(2018-WSW-003)


Evaluation of the effects of deoxycholic acid on gastric microbiota in INS ⁃ GAS mouse model of intestinal type gastric cancer based on 16S rDNA amplicon sequencing
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    摘要:

    目的:探究次级胆汁酸脱氧胆酸(deoxycholic acid,DCA)对肠型胃癌模型INS-GAS小鼠胃内菌群结构的影响。方法:将10只8周龄雄性INS-GAS小鼠随机分为2组,每组5只,一组予以0.2% DCA饮水,另一组饮用生理盐水(normal saline, NS)溶剂。另有5只同窝雄性FVB/N野生型小鼠予以0.2% DCA饮水作为基因型对照组。3个月饮水干预结束后收集各组小鼠胃内容物行16S rDNA扩增子测序,进行胃内菌群相对丰度、α多样性指数、β多样性及差异物种分析。分离胃体和胃窦黏膜组织,qRT-PCR检测肠化标志物cdx2、klf5、vil1和muc2 mRNA表达。结果:与对照组小鼠相比,DCA改变了INS-GAS小鼠胃内优势菌种,属水平上相对丰度最高的菌种由乳杆菌科菌属(61.93%)变为未定义蓝藻细菌属(58.70%)。两组小鼠胃内菌群丰度及多样性均无显著差异(ACE指数P=0.213,Chao1指数P=0.280;Shannon指数P=0.391,Simpson指数P=0.205)。主成分分析发现两组小鼠胃内菌群结构差异度大,组间差异大于组内差异(R=0.368,P=0.032)。DCA饮水促进INS-GAS小鼠胃内蓝藻细菌在门、纲、目、科和属各个水平上显著增高,毛螺菌科Anaerostipes菌属以及两种蓝藻细菌属菌种Lolium perennePhaseolus vulgaris丰度也显著增高。此外,DCA饮水同样导致INS-GAS小鼠胃内厚壁菌门、杆菌纲和乳杆菌(目、科和属水平)丰度显著降低,罗伊氏乳杆菌丰度也显著降低。DCA饮水的INS-GAS小鼠胃体及胃窦部黏膜组织muc2 mRNA表达均显著高于NS饮水的INS-GAS小鼠(P均< 0.05)。此外,同为DCA饮水的INS-GAS小鼠胃内菌群丰度显著高于FVB/N小鼠(ACE指数P=0.022; Chao1指数P=0.028),两组小鼠组内差异大于组间差异,但尚无统计学意义(R=-0.056,P=0.647)。结论:DCA改变了INS-GAS 小鼠胃内菌群结构,主要为蓝藻细菌属和毛螺菌科Anaerostipes菌属相对丰度增高,乳杆菌属相对丰度降低,且促进了胃黏膜肠化标志物muc2表达。

    Abstract:

    Objective:We aimed to investigate the effects of a secondary bile acid deoxycholic acid(DCA)on the gastric microbial community in INS-GAS(Insulin-Gastrin)mice model of intestinal type gastric cancer. Methods:Ten 8-week-old male INS-GAS mice were randomly divided into two groups,one group was given 0.2% DCA drinking water,and the other group was given normal saline (NS)solvent. Another 5 male FVB/N wild -type mice in the same litter were given 0.2% DCA as a genotype control group. After 3 months of drinking water intervention,the gastric contents of each group were collected for 16S rDNA amplicon sequencing,and the relative abundance of gastric microbiota,α diversity,β diversity and significantly different species were analyzed. The gastric body and antrum mucosal tissues were isolated respectively,and the mRNA expressions of intestinal markers cdx2,klf5,vil1 and muc2 were detected by qRT-PCR. Results:Compared with the NS group,DCA treatment changed the dominant bacteria in the stomachs of INS- GAS mice,and the bacteria with the highest relative abundance on the level of genus changed from the Lactobacillus(61.93%)to the undefined Cyanobacteria genus(58.70%). There were no remarkably differences in the abundance and diversity of gastric microflora between the two groups(ACE index P=0.213,Chaol index P=0.280;Shannon index P=0.391,Simpson index P=0.205). Principal component analysis found that the two groups had a considerable difference in the structure of the gastric microbiota,and the difference between the two groups was greater than the difference within the groups(R=0.368,P=0.032). DCA treatment significantly increased the abundance of Cyanobacteria in the stomach of INS -GAS mice at the levels of phylum,class,order,family and genus,and also upregulated the abundance of the Anaerostipes genus and the Cyanobacteria genus Lolium perenne and Phaseolus vulgaris. In addition, significantly higher enrichment of Lactobacillus including Lactobacillus reuteri,belonging to the phylum Firmicutes was found in gastric contents of INS -GAS mice drinking DCA compared with the control group. The expression of muc2 mRNA in the gastric body and antrum mucosa of INS-GAS mice drinking DCA was considerably higher than that of INS-GAS mice drinking NS(both P < 0.05). The abundance of gastric microbiota in the stomach of INS-GAS mice with the same DCA intervention was significantly higher than that of FVB/N mice(ACE index P=0.022;Chao1 index P=0.028). However,INS-GAS(DCA)and FVB/N(DCA)mice had greater differences within groups than between groups(R=-0.056,P=0.647). Conclusion:DCA intake changed the structure of the gastric microbiota of INS-GAS mice. The relative abundances of Cyanobacteria and Anaerossipes were significantly increased,while the relative abundance of Lactobacillus decreased,and promoted the expression of Muc2 mRNA.

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金多晨,黄可婷,徐妙,党旖旎,张国新.基于16SrDNA扩增子测序探究脱氧胆酸对肠型胃癌模型INS⁃GAS小鼠胃内菌群结构的影响[J].南京医科大学学报(自然科学版),2022,42(12):1651-1657

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  • 在线发布日期: 2022-12-12
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