高通量测序分析结肠腺瘤患者肠道黏膜菌群的构成变化

High-throughput sequencing for analysis of structural change of intestinal microbiota in patients with colorectal adenoma

目的探讨结直肠癌发展早期肠道菌群的变化规律。方法通过结肠镜活检收集31例结肠腺瘤患者的腺瘤组织以及20例健康志愿者的肠道黏膜,提取基因组DNA,对细菌16SrRNA基因序列V_3~V_4高变区进行PCR扩增,lluminaMiSeq平台上高通量测序分析。结果结肠腺瘤患者的肠道黏膜菌群多样性比健康对照人群显著增高(P<0.01),腺瘤组黏膜菌群结构和健康对照者相比差异显著。门水平上:结肠腺瘤组变形菌门明显增多(P<0.01),厚壁菌门/拟杆菌门比值下降。属水平上:结肠腺瘤组乳球菌属、链球菌属和假单胞菌属增多(P<0.01),而肠球菌属,杆菌属丰度明显下降(P<0.01)。不同病理类型腺瘤比较发现:结肠高级别和低级别上皮内瘤变组肠道黏膜菌群整体结构相似,埃希菌-志贺菌属在高级别上皮内瘤变组呈现上升趋势,但差异无统计学意义(P>0.05)。结论结肠腺瘤患者肠道菌群多样性和构成与正常健康人存在明显差异,腺瘤患者黏膜菌群结构发生明显失衡,形成了易于肿瘤生长的肠道微生态环境。

Objective To investigate the taxonomic richness and diversity of gut microbiota in patients with colorectal adenoma and elucidate the role of gut microorganisms in precancerous lesions in the colon and rectum.Method Adenomatous tissues from 31 patients with colorectal adenoma and normal intestinal mucosal tissues from 20 healthy control subjects were collected through colonoscopy.The total bacterial genomic DNA was extracted,and the V_3-V_4 hypervariable region in bacterial 16 S r RNA gene was amplified using polymerase chain reaction and sequenced on an Illumina Mi Seq platform.Results Patients with colorectal adenomas had a higher alpha diversity and richness indices compared to the healthy controls （P〈0.01）.The mucosal microbiota in colorectal adenoma tissue showed a distinctive structural difference from that in normal intestinal mucosal tissues.At the phylum level,a large decrease in Firmicutes with concomitant relative expansion of Proteobacteria was observed in patients with colorectal adenomas,resulting in a significant decrease in the Firmicutes/Bacteroidetes ratio （P〈0.01）.At the genus level,Lactococcus and Pseudomonas were enriched whereas Enterococcus,Bacillus,and Solibacillus were reduced obviously in the preneoplastic tissues （P〈0.01）.We also found a similar gut microbiome composition between low-grade and high-grade intraepithelial neoplasia; the ratio of Escherichia-Shigella tended to increase in high-grade intraepithelial neoplasia,but this change was not statistically significant （P=0.28）.Conclusion Significant changes in the structure of the intestinal flora occur in patients with colorectal adenomas,indicating that the association of dysbiosis of the gut microbiota with the occurrence of a pro-oncogenic microenvironment.