慢性HBV感染者肠道真菌菌群生态结构研究

目的 探讨慢性HBV感染者肠道真菌菌群的分子生态结构变化特点.方法 采用真菌18S rDNA通用引物对乙肝肝硬化患者、慢性乙型肝炎患者、HBV携带者和健康志愿者4组研究对象的粪便标本DNA进行PCR扩增,扩增产物进行18S rRNA基因克隆,构建真菌18S rDNA克隆文库,利用限制性片段长度多态性(RFLP)技术筛选阳性克隆子并进行测序,绘制系统发育树,获取各组研究对象肠道真菌菌群结构特征.结果 所有阳性克隆子经过酶切分析和测序,共获得29个操作分类单元(OTUs),归属于3个真菌类群:接合菌纲(3.4%)、子蓑菌纲(82.8%)和担子菌纲(13.8%),其中主要优势菌属为念珠菌属(Candida spp.)、未能培养真菌(uncultured fungus)、酵母菌属(Saccharomyces spp.),分别占克隆文库的29.2%、15.9%、15.0%.乙肝肝硬化患者组、慢性乙型肝炎患者组、HBV携带者组和健康志愿者组的肠道真菌菌群分别存在有20、16、12、14个OTUs.结论 人类肠道中存在较为丰富的真菌类群,慢性HBV感染者肠道真菌菌群分子生态结构发生明显改变,提示肠道真菌菌群生态结构改变与乙肝发展历程相关.

Molecular ecological structure of intestinal fungal flora in patients with chronic hepatitis B infection

Objective To investigate the ecological structure of intestinal fungal flora of patients with chronic hepatitis B infection. Methods HBV-liver cirrhosis patients, chronic hepatitis B patients, HBV carriers, and healthy volunteers were selected as research subjects to extract the total DNA from stool of each subject, 18S rRNA genes of intestinal fungi was cloned and sequence was analyzed for the construction of 18S rDNA clone libraries and phylogenetic tree, diversity and structural characteristics of intestinal fungal flora in each group was then analyzed. Results A total of 29 operational taxonomic units (OTUs) of intestinal fungal flora in all research subjects was acquired. All positive clones belonged to three fungal taxa: Zygomycetes (3.4%) , Ascomycetes (82. 8%) and Basidiomycetes (13. 8%) . The dominant fungal community was Candida spp. , uncultured fungus and Saccharomyces spp. , which accounted for 29. 2% , 15. 9%, 15.0% respectively. The OTUs which belonged to HBV-liver cirrhosis patients, chronic hepatitis B patients, HBV carriers, and healthy volunteers was 20,16,12,14 respectively. Conclusion There is an abundant fungal flora in the human intestine. Ecological structure of intestinal fungal flora appears to be various in patients with chronic hepatitis B infection, which indicate the close relationship between the alteration of ecological structure of fungal flora and the stage of chronic hepatitis B infection.