Illumina高通量测序分析人结肠恶变组织黏膜菌群变化

来源 :中国热带医学 | 被引量 : 0次 | 上传用户:cyuch
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目的分析结肠腺瘤及腺癌患者的正常组织和恶变组织黏膜菌群特征,试图寻找癌变过程中黏膜菌群变化趋势。方法研究对象为广东医学院附属南山医院消化内科就诊的5例腺瘤和2例腺癌患者,肠镜下取微量的结肠黏膜组织,每例患者收集病变组织和病变旁5~10 cm处的正常组织,用试剂盒提取基因组DNA,PCR扩增16S r RNAV3~V4区,对PCR产物进行二代Illumina高通量测序,通过COPE软件分析和统计样品序列数目,在0.97相似度下利用QIIME(v1.8.0)软件将序列聚类为用于物种分类的OTUs(Operational taxonomic units),进一步分析结肠正常组织和恶变组织的黏膜菌群物种丰度及结构组成特点。结果 14个样本共得到3 306个OTUs,测序深度不一;腺癌患者结肠正常组织黏膜菌群多样性指数高于恶变组织,腺瘤患者的两组指数比较没有一致规律。14个样本的共有菌门为6个,7例患者的黏膜菌群均以厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)以及变形菌门(Proteobacteria)为主,在2例腺癌患者恶变组织的黏膜菌群中,梭杆菌门(Fusobacteria)比正常组织显著增多,占30%以上,成为了优势菌门,而在5例腺瘤患者的两组黏膜菌群比较中均没有此现象。在纲和属的分类水平上,梭杆菌纲(Fusobacteriia)和梭杆菌属(Fusobacterium)仍然在腺癌患者恶变组织和正常组织中存在显著性差异,均明显增多且成为黏膜定植的优势菌。结论腺瘤到腺癌演变过程中,黏膜定植菌群的多样性变化未出现明显趋势,但根据梭杆菌的变化特点推测其可能是因癌症发生发展导致了数量的增多,而并非是结肠癌发生的病原学因子。 Objective To analyze the characteristics of mucosal flora in normal tissues and malignant tissues of patients with colon adenomas and adenocarcinomas, and try to find out the trend of mucosal flora in the course of carcinogenesis. Methods A total of 5 adenomas and 2 adenocarcinoma patients attending the Department of Gastroenterology of Nanshan Hospital, Guangdong Medical College were enrolled. The colonic mucosa was collected under colonoscopy. The lesions were collected at 5 to 10 cm in each patient. In normal tissues, genomic DNA was extracted using the kit, 16S r RNAV3~V4 regions were amplified by PCR, Illumina high-throughput sequencing was performed on the PCR products, COPE software was used to analyze and count the number of sample sequences, and QIIME was used at a similarity of 0.97 ( V1.8.0) The software clusters the sequences into OTUs (Operational taxonomic units) for species taxonomy to further analyze the species abundance and structural composition of the mucosal flora of normal and malignant colon tissue. Results A total of 3 306 OTUs were obtained in 14 samples with different sequencing depths. The mucosal diversity index of colonic normal tissues in patients with adenocarcinoma was higher than that in malignant tissues. There was no consistent pattern in the two groups of adenomas. There were 6 common bacteria in 14 samples. The mucosal flora of 7 patients were mainly Firmicutes, Bacteroidetes, and Proteobacteria. In 2 patients with adenocarcinoma In the mucosal flora of malignancies, Fusobacteria significantly increased compared with normal tissues, accounting for more than 30%, becoming a dominant phylum, and this phenomenon was not found in the two groups of mucosal flora in 5 patients with adenoma. . At the classification level of genera and genus, Fusobacteriia and Fusobacterium still had significant differences in the malignant tissues and normal tissues of patients with adenocarcinoma, both of which significantly increased and became dominant bacteria colonization. Conclusion During the evolution of adenocarcinoma to adenocarcinoma, the diversity of mucosal colonization did not show a clear trend. However, according to the characteristics of Fusobacterium, it may be due to the development of cancer, resulting in an increase in the number, rather than colon cancer. Etiological factors.
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