基于高通量测序的营养不良儿童肠道菌群多样性研究

Diverse research of intestinal flora in children with malnourished based on high-throughput sequence

目的 采用高通量测序方法,比较营养不良儿童与正常儿童肠道菌群物种多样性及组成特点的差异。方法 2017年1月至6月,在广西壮族自治区妇幼保健院儿童营养门诊就诊的营养不良患儿中,纳入20例为研究对象;同时纳入正常健康儿童20例为对照组。采集粪便样本,提取细菌DNA,通过两步PCR的方法构建文库,扩增16SrDNA中V4-V5区进行高通量测序。进行生物信息学分析,构建稀释性曲线;采用mothur软件对Alpha多样性进行计算。利用R语言进行Heatmap分析、主成分分析PCA(PrincipalComponentAnalysis)、典型关联分析CCA(CanonicalCorrelationAnalysis)/冗余分析RDA(RedundancyAnalysis),分别在样本的门和属两个分类水平上进行数据整合归类比较统计。结果 营养不良组与对照组儿童的月龄、性别、分娩方式、出生胎龄以及喂养等基本情况比较,差异均无统计学意义。两组儿童肠道菌群的丰富度和均匀度比较,差异无统计学意义。两组儿童肠道菌群门水平微生物共检出14种,营养不良组的硬壁菌门(Firmicutes)及未分类菌的OUT数低于对照组,拟杆菌(Bacteroidetes)的OUT数高于对照组(P<0.05)。两组儿童肠道菌群属水平微生物共检出154种,其中多形杆状菌(Bacteroides)、乳酸菌(Lactobacillus)、罗斯菌(Roseburia)等24个属的OTU在两组间差异具有统计学意义(P<0.05),其中营养不良组的多型杆状菌(Bacteroides)和罗斯菌(Roseburia)的OUT数高于对照组,乳酸菌(Lactobacillus)的OUT数低于对照组。将两组样本进行主成分PCA分析,根据样本聚类情况,营养不良组与对照组虽然距离相近,但仍可看出两组分别聚集。结论 营养不良儿童肠道菌群在门水平中硬壁菌偏低,在属水平中多形杆状菌及罗斯菌含量偏高,因此可根据不同菌属的作用特点来调整菌群,以期达到菌群平衡、吸收良好、体格发育正常的良性循环。

Objective To compare the differences between malnourished children and normal children in diversity of intestinal flora and character of composition by high-throughput sequence. Methods From January to June 2017, 20 malnourished children who were treated in the Child Healthcare Department of The Maternal Child Health Hospital of Guangxi Zhuang Autonomous Region were recruited. Meanwhile 20 healthy children were selected as control group. Fecal specimens were collected and bacterial DNA was extracted. The library was constructed through two-step PCR, and the V4/V5 region of 16S rDNA was amplified with high-throughput sequence. The results were analyzed by bioinformatics and the dilution curve was constructed. Alpha diversity was counted by mothur software. Heatmap analysis, Principal Component Analysis (PCA), Canonical Correlation Analysis (CCA)/Redundancy Analysis (RDA) were analysed by R language. Data integration and classification were carried out at the level of phylum and genus species respectively. Results There was no statistical significance between malnutrition group and control group in terms of age, sex, mode of delivery, gestational age, and feeding. There was no statistical significance in the species richness and evenness between the two groups. A total of 14 microorganisms for phylum were detected in intestinal flora from two groups, the number of OTU for Firmicutes and unclassified bacteria in malnutrition group was lower than that in control group, the number of OTU for Bacteroidetes in malnutrition group was higher than that in control group(P<0.05). A total of 154 microorganisms for genus were detected in intestinal flora in two groups, the OTUs of 24 genera, including Bacteroides, Lactobacillus and Roseburia, had statistical significance between the two groups (P<0.05). The number of OTU for Bacteroides and Roseburia in malnutrition group was higher than that in control group, the number of OTU for Lactobacillus in malnutrition group was lower than that in control group. PCA analysis of samples in two groups showed that although it was close between malnutrition group and control group in distance , they were still clustered separately. Conclusions Malnourished children have lower contents of Firmicutes in phylum species, and higher contents of Bacteroides and Roseburia in genus species. The children's intestinal flora can be adjusted according to the characteristics of different bacteria to achieve the balance of bacteria, well absorption, a virtuous cycle of normal physical development.