高通量测序技术分析壳寡糖对小鼠肠道菌群的影响

EFFECT OF CHITOSAN OLIGOSACCHARIDE ON INTESTINAL FLORA IN MICE USING ILLUMINA-MISEQ HIGH-THROUGHPUT SEQUENCING PLATFORM

目的研究壳寡糖对小鼠肠道菌群的影响。方法利用诺氟沙星灌胃建立肠道微生态失调小鼠模型,然后用壳寡糖对昆明小鼠每天进行灌胃,连续灌胃14d后,在无菌条件下采集各组小鼠盲肠内容物,提取基因组DNA,对样本的16S rDNA基因V3-V4区进行PCR扩增,采用Illumina-Miseq高通量测序平台测序,并对各样本进行生物信息学分析。结果体外实验结果显示,壳寡糖能显著促进乳杆菌的生长,抑制大肠杆菌的生长,且呈浓度剂量依赖。OTUs结果显示,利用抗生素处理后,小鼠肠道菌群的数量与对照组相比明显减少,但壳寡糖能增加和恢复小鼠肠道内的菌群数量。肠道菌群Alpha多样性和Beta多样性分析结果显示,壳寡糖能增加小鼠肠道菌群的丰富度和多样性,可恢复抗生素对小鼠肠道菌群平衡造成的破坏。在属水平上对样品进行菌群种类和丰度分析的结果显示,4组样本在菌群种类和丰度上均存在较大的差异。Azospira、Bacteroides(拟杆菌属)、Lactobacillus(乳杆菌属)和Alistipes(另枝菌属)在4组样本中的菌属序列数目变化最为显著。其中壳寡糖组能够抑制有害菌Azospira的生长和繁殖,提高肠道益生菌群Bacteroides、Lactobacillus和Alistipes的种类和丰度,而自然恢复组与之相反。结论壳寡糖能够提高小鼠肠道益生菌群的种类和丰度,可对抗生素造成的肠道菌群紊乱具有显著的改善和恢复作用。

Objective To study the effect of oligochitosan on intestinal flora in mice by intragastric administration of norfloxacin.Methods A mouse model of intestinal microecology was established by intragastric administration of norfioxacin and then administered daily with chitosan oligosaccharide in Kunming mice. After 14d administration, the cecum contents were collected under sterile conditions to extract genomic DNA samples and the 16S rDNA V3-V4 genes were analysed by PCR, Illumina -Miseq high-throughput sequencing platform, and bioinformatics. Results In vitro, chitosan oligosaccharide could significantly promote the growth of Lactobacillus and inhibit the growth of E. coli, in a dose and dose dependent manner. The results of OTUs showed that the number of intestinal microflora in mice was decreased significantly after treatment with antibiotics, but chitosan oligosaccharide could recover the number of intestinal flora in mice. The results of Alpha diversity analysis showed that chitosan oligosaccharide could increase the abundance and diversity of intestinal flora in mice. Beta diversity analysis showed that chitosan oligosaccharide could restore the damage caused by antibiotics to intestinal flora balance in mice. There were significant differences in Genus richness between the 4 samples. Among them, ,4zospira, Bacteroides, Lactobacillus and .4listipes（other bacteria） had the most significant sequence variation in 4 samples. The chitosan oligosaccharide could inhibit the growth and reproduction of harmful bacteria Azospira, improve intestinal probiotics group Bacteroides, Lactobacillus and Alistipes, abundance. Conclusion Chitosan oligosaccharide can improve the probiotic species and abundance of intestinal flora in mice, and restore the intestinal flora disorder caused by antibiotics.