患2型糖尿病的肥胖人群肠道菌群分析

doi:10.3969/j.issn.1673-8640.2016.010.003

摘要/Abstract

摘要：

目的探讨患2型糖尿病的肥胖人群肠道菌群的变化特征。方法以21例患2型糖尿病的肥胖者作为研究组,21名血糖正常的肥胖志愿者作为对照组。取研究组和对照组新鲜粪便,采用Illumina Miseq高通量测序平台对粪便样本中所有细菌的16S rRNA-V3区进行DNA测序,分析肠道菌群物种的丰度和分布,并进行聚类分析。采用实时定量聚合酶链反应（PCR）对肠道菌群进行定量分析。结果成功进行了DNA测序分析,样本稀疏曲线显示测序深度充分,测序覆盖深度（Coverage指数）>0.99。研究组肠道菌群2个丰度指数（Ace和Chao1）均低于对照组（P<0.05）,2个多样性指数（Shannon和Simpson）分别低于和高于对照组（P<0.05）。实时定量PCR结果显示,在门水平,研究组的厚壁菌门和放线菌门均低于对照组（P<0.05）,而拟杆菌门高于对照组（P<0.05）,其他2个菌门（变形杆菌门和梭形杆菌门）差异无统计学意义（P>0.05）;在属水平,研究组的韦荣球菌属、Ⅳ型梭菌属和拟杆菌属均高于对照组（P<0.05）,而乳杆菌属、Blautia球菌-直肠真杆菌属、普雷沃菌属和双歧杆菌属均低于对照组（P<0.05）。结论患2型糖尿病的肥胖人群肠道菌群的丰度和多样性下降,定量分析肠道菌群的构成对该人群有特殊意义。

关键词: 肠道菌群, 2型糖尿病, 肥胖, 高通量测序

Abstract:

Objective To investigate the characteristics of gut microbiota in obese population with type 2 diabetes mellitus. Methods A total of 21 obese patients with type 2 diabetes mellitus（study group） and 21 obese subjects with normal glucose（control group） were enrolled. The fecal samples were collected and determined by Illumina Miseq high-throughput sequencing for the V3 region of 16S rRNA gene. The abundance and distribution were analyzed by cluster analysis. Gut microbiota was analyzed by real-time quantitation polymerase chain reaction（PCR）.Results The analysis of DNA sequence was successfully performed. The rare faction curves showed that adequate sequencing depth was achieved,and the coverage indices were >0.99. The Ace and Chao1 indices of study group were lower than those of control group（P<0.05）. The Shannon index or Simpson index of study group was lower or higher than that of control group（P<0.05）. The results of real-time quantitation PCR showed that the quantities of Actinobacteria and Firmicutes were lower（P<0.05）,while that of Bacteroidetes was higher in study group than that in control group（P<0.05）. The quantities of Proteobacteria and Fusobacteria had no statistical significance between the 2 groups（P>0.05）. The quantities ofVeillonella,Clostridium Ⅳ and Bacteroides were higher,and the quantities of Lactobacillus,Blautia coccoides-Eubacterium rectale,Prevotella and Bifidobacterium were lower in study group compared with those in control group（P<0.05）. Conclusions The obese population with type 2 diabetes mellitus has low abundance and diversity of gut microbiota. The quantitation analysis of the composition of gut microbiota has a certain significance.

Key words: Gut microbiota, Type 2 diabetes mellitus, Obese, High-throughput sequencing

中图分类号:

R446.5

曾艺鹏, 胡燕, 吴平, 冯新格, 谷成英, 郭亚芳. 患2型糖尿病的肥胖人群肠道菌群分析[J]. 检验医学, 2016, 31(10): 848-853.

ZENG Yipeng, HU Yan, WU Ping, FENG Xin'ge, GU Chengying, GUO Yafang. Analysis of gut microbiota in obese population with type 2 diabetes mellitus[J]. Laboratory Medicine, 2016, 31(10): 848-853.

图/表 7

参考文献 13

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菌种	引物序列	产物序列长度（bp）
拟杆菌门	F: CATGTGGTTTAATTCGATGAT	126
拟杆菌门	R: AGCTGACGACAACCATGCAG
厚壁菌门	F: ATGTGGTTTAATTCGAAGCA	126
厚壁菌门	R: AGCTGACGACAACCATGCAC
放线菌门	F: CGCGGCCTATCAGCTTGTTG	600
放线菌门	R: CCGTACTCCCCAGGCGGGG
变形杆菌门	F: CATGACGTTACCCGCAGAAGAAG	195
变形杆菌门	R: CTCTACGAGACTCAAGCTTGC
梭形杆菌门	F: CCCTTCAGTGCCGCAGT	273
梭形杆菌门	R: GTCGCAGGATGTCAAGAC
拟杆菌属	F: GAGAGGAAGGTCCCCCAC	106
拟杆菌属	R: CGCTACTTGGCTGGTTCAG
普雷沃菌属	F: GGTTCTGAGAGGAAGGTCCCC	121
普雷沃菌属	R: TCCTGCACGCTACTTGGCTG
Ⅳ型梭菌属	F: GCACAAGCAGTGGAGT	239
Ⅳ型梭菌属	R: CTTCCTCCGTTTTGTCAA
乳杆菌属	F: GAGGCAGCAGTAGGGAATCTTC	126
乳杆菌属	R: GGCCAGTTACTACCTCTATCCTTCTTC
韦荣球菌属	F: ACCAACCTGCCCTTCAGA	343
韦荣球菌属	R: CGTCCCGATTAACAGAGCTT
Blautia球菌-直肠真杆菌属	F: CGGTACCTGACTAAGAAGC	429
Blautia球菌-直肠真杆菌属	R: AGTTTCATTCTTGCGAACG
双歧杆菌属	F: CTCCTGGAAACGGGTGG	550
双歧杆菌属	R: GGTGTTCTTCCCGATATCTACA

菌种	引物序列	产物序列长度（bp）
拟杆菌门	F: CATGTGGTTTAATTCGATGAT	126
拟杆菌门	R: AGCTGACGACAACCATGCAG
厚壁菌门	F: ATGTGGTTTAATTCGAAGCA	126
厚壁菌门	R: AGCTGACGACAACCATGCAC
放线菌门	F: CGCGGCCTATCAGCTTGTTG	600
放线菌门	R: CCGTACTCCCCAGGCGGGG
变形杆菌门	F: CATGACGTTACCCGCAGAAGAAG	195
变形杆菌门	R: CTCTACGAGACTCAAGCTTGC
梭形杆菌门	F: CCCTTCAGTGCCGCAGT	273
梭形杆菌门	R: GTCGCAGGATGTCAAGAC
拟杆菌属	F: GAGAGGAAGGTCCCCCAC	106
拟杆菌属	R: CGCTACTTGGCTGGTTCAG
普雷沃菌属	F: GGTTCTGAGAGGAAGGTCCCC	121
普雷沃菌属	R: TCCTGCACGCTACTTGGCTG
Ⅳ型梭菌属	F: GCACAAGCAGTGGAGT	239
Ⅳ型梭菌属	R: CTTCCTCCGTTTTGTCAA
乳杆菌属	F: GAGGCAGCAGTAGGGAATCTTC	126
乳杆菌属	R: GGCCAGTTACTACCTCTATCCTTCTTC
韦荣球菌属	F: ACCAACCTGCCCTTCAGA	343
韦荣球菌属	R: CGTCCCGATTAACAGAGCTT
Blautia球菌-直肠真杆菌属	F: CGGTACCTGACTAAGAAGC	429
Blautia球菌-直肠真杆菌属	R: AGTTTCATTCTTGCGAACG
双歧杆菌属	F: CTCCTGGAAACGGGTGG	550
双歧杆菌属	R: GGTGTTCTTCCCGATATCTACA

组别	年龄（岁）	性别（男/女）	BMI （kg/m²）	空腹血糖(mmol/L)	糖化血红蛋白（%）	空腹胰岛素（mU/L)	甘油三酯(mmol/L)	总胆固醇(mmol/L)
对照组	52.11±8.56	11/10	30.95±2.77	12.16±3.13	10.71±2.31	15.26±6.17	2.13±1.32	5.39±1.32
研究组	48.12±9.33	13/8	31.23±2.91	5.29±1.19	4.22±1.23	9.35±3.09	2.09±1.38	5.53±1.21
P值	0.16	0.49	0.75	0.00	0.00	0.00	0.92	0.73

组别	年龄（岁）	性别（男/女）	BMI （kg/m²）	空腹血糖(mmol/L)	糖化血红蛋白（%）	空腹胰岛素（mU/L)	甘油三酯(mmol/L)	总胆固醇(mmol/L)
对照组	52.11±8.56	11/10	30.95±2.77	12.16±3.13	10.71±2.31	15.26±6.17	2.13±1.32	5.39±1.32
研究组	48.12±9.33	13/8	31.23±2.91	5.29±1.19	4.22±1.23	9.35±3.09	2.09±1.38	5.53±1.21
P值	0.16	0.49	0.75	0.00	0.00	0.00	0.92	0.73

组别	Reads指数	Coverage指数	OTU	Ace	Chao1	Shannon	Simpson
对照组	23 454.33±2 224.89	0.998 1±0.002 1	188.12±19.16	209.77±32.21	225.54±43.55	3.23±0.89	0.088 7±0.050 1
研究组	22 921.45±1 258.76	0.997 2±0.001 8	113.44±17.25	119.09±24.91	187.49±36.42	2.39±0.76	0.128 7±0.071 2
P值	0.057 6	0.143 8	0.000 0	0.000 0	0.003 8	0.002 1	0.041 5