基于高通量测序的营养不良儿童肠道菌群多样性研究

·论著·

基于高通量测序的营养不良儿童肠道

菌群多样性研究

吴薇岚　江蕙芸（广西壮族自治区妇幼保健院　儿童保健科，广西　南宁

530000）

【摘要】 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16S rDNA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（Principal Component Analysis）、典型关联分析CCA（Canonical Correlation Analysis）/冗

余分析RDA（Redundancy Analysis)，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。

【关键词】营养不良儿童；高通量测序；硬壁菌门；多形杆状菌；罗斯菌

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

sequence

WU Wei-lan, JIANG Hui-yun (Child Healthcare Department, The Maternal Child Health Hospital of

Guangxi Zhuang Autonomous Region, Guangxi, Nanning 530000, China)

Corresponding author: JIANG Hui-yun (Email: 170130813@ )

【Abstract】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

DOI：10.3969/j.issn.2095-5340.2019.03.006

基金项目：广西医疗卫生适宜技术开发与推广应用项目（S201616）

通讯作者：江蕙芸（Email：170130813@ qq. com）

营养不良是指由于各种营养物质摄入过量或不足、吸收障碍或消耗过度所致的一种营养失衡症。营养不良是一个全球性的重大问题，在欧美等发达国家以肥胖常见，在亚洲、非洲等发展中国家则是以低体重、消瘦、生长迟缓多见。在南亚和东非少数国家5岁以下低体重儿童中，分别有33%和28%的患儿营养不良与肠道通透性增加、腹泻、吸收障碍和免疫功能低下、肠炎相关［1-2］，这些因素均严重影响着学龄前儿童的健康成长［3-4］。迄今为止，营养素干预、饮食调整及行为疗法等解决方案尚未能有效治疗营养不良。在人类的生长发育和疾病发生过程中，肠道微生物群落常常被看做是人类的“第二基因组”［5］，在人类健康中起着非常重要的作用。有研究显示，肠道菌群对宿主从食物中提取营养物质起着重要作用，特别是对儿童营养及生长发育过程影响深刻［6］。宿主肠道菌群的组成结构及丰度可以调控食物中营养物质的吸收和热量的供给过程，如果肠道微生态平衡发生严重紊乱，即菌群失调，便可影响宿主对营养和能量的获取［7-8］。如结肠内的厌氧菌可以对未消化的食物进行化学作用，参与多种婴幼儿生长发育所需营养物质（蛋白质、氨基酸、糖、脂类、维生素等）的合成与代谢，由此可见，探讨营养不良儿童肠道菌群的变化具有重要意义。本研究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，探讨儿童营养不良与肠道微生物菌群构成的相关性。

1　资料与方法

1.1　研究对象　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（A组），其中男性8例，女性12例，平均月龄为（18±15）个月（4～48个月）。纳入标准：营养不良原因均为喂养和饮食因素引起，如挑食、偏食、厌食、缺乏科学喂养方式等。排除标准：近半年有感染性腹泻、抗生素及益生菌使用史，先天性消化道畸形、消化道手术史。同时纳入健康儿童20例为对照组（B组），其中男性9例，女性11例，平均月龄为（22±12）个月（4～50个月）。

1.2　诊断标准　儿童营养不良诊断标准参考美国国家健康统计中心（CNHS）制定的国际标准（即

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.

【Key words】Malnourished child; High throughput sequence; Firmicutes; Bacteroides; Roseburia