【作者】 陈燕飞；

【导师】 李兰娟；

【作者基本信息】 浙江大学 ， 内科学， 2013， 博士

【副题名】从结构到功能

【摘要】 肝硬化是我国消化系统常见疾病,患者常死于如上消化道出血、自发性细菌性腹膜炎等各种并发症。肠道与肝脏通过“肠-肝轴”密切关联。肝硬化时肠道微环境常因肝脏功能损害和门脉高压受到直接和间接的影响。研究表明肠道细菌移位参与肝硬化各种并发症的发生发展。肝硬化状态下肠道细菌移位发生主要与三方面因素有关：肠道细菌过度生长、肠壁通透性增加以及机体免疫力下降。以往对于肝硬化肠道菌群结构研究多依赖细菌培养法。然而很多肠道细菌无法在体外分离培养,因此无法从整体对肠道菌群结构进行分析。近年来,越来越多的肠道菌群研究依赖非培养的分子生态学方法进行,这些方法能够更好的反映肠道菌群群落多样性特点,为理解肠道菌群在肝硬化病理机制中的作用提供依据。为了全面分析肝硬化肠道微生态失衡情况,本研究拟利用分子生态学方法对肝硬化患者肠道菌群进行结构和功能两方面研究。方法本研究以酒精性肝硬化患者、乙肝肝硬化患者及健康对照个体为研究对象,利用基于16S rRNA的454高通量测序和荧光定量PCR法分析患者肠道菌群群落结构特点,同时利用微生物功能基因芯片GeoChip分析患者肠道微生物功能基因多样性,从结构到功能对肝硬化患者肠道微生态进行全面分析,并探讨肝硬化不同病因对肠道菌群的影响。结果1.从群落结构上来看,肝硬化患者粪便菌群结构与健康对照存在明显差异。在门的水平上,肝硬化患者肠道菌群中类杆菌门细菌比例明显下降,而变形菌门和梭杆菌门细菌比例显著增加。在科的水平上,与健康对照组相比,肝硬化患者肠道菌群中肠杆菌科、韦荣球菌科和链球菌科细菌明显增加,毛螺菌科细菌比例明显下降。肠道链球菌科细菌比例与肝硬化Child-Pugh评分呈正相关,毛螺菌科细菌比例与Child-Pugh评分呈负相关。链球菌等潜在致病菌在肝硬化患者肠道中显著增加,可能与肝硬化并发感染等密切相关。2.从功能结构看,肝硬化患者肠道菌群涉及营养物质代谢通路的功能基因显著缺失,如氨基酸合成和代谢基因,脂类代谢基因、辅因子合成代谢基因等较正常对照组明显减少。肠道菌群营养物质分解潜力下降可能与肝硬化营养不良发生相关。3.在群落结构上,酒精性肝硬化与乙肝肝硬化患者相比差异较小,主要表现为酒精性肝硬化患者普雷沃菌科细菌比例显著增加。在功能结构上,酒精性肝硬化与乙肝肝硬化患者相比差异显著,酒精性肝硬化肠道微生物富集了很多涉及重金属降解、有机污染物降解、应激反应、毒力等通路的功能基因。因此长期大量饮酒可能主要在功能层面对菌群产生影响。结论肝硬化患者菌群群落结构及功能结构都发生了显著改变,菌群改变可能与肝硬化并发感染、营养不良及肝性脑病等并发症有关。本研究对肝硬化患者肠道微生态失衡进行了从结构到功能的全面分析,可以为临床通过菌群调节诊治肝硬化提供理论依据。更多还原

【Abstract】 Liver cirrhosis is the pathologic end stage of chronic liver disease. In China and some other Asian countries, hepatitis B virus infection is one of the leading causes. On the other hand, alcohol-related cirrhosis is a major cause of morbidity and mortality in Western countries. Increasing evidence suggests that gut flora is implicated in the pathogenesis of liver cirrhosis complications. It is well recognized that the gut flora may play an important role in the development of complications of liver cirrhosis, including bacterial infections, the hyperdynamic circulatory state, and hepatic encephalopathy. Although it is widely accepted that cirrhosis leads to characteristic changes in intestinal microbiota, this has not been easily demonstrated in previous studies using culture-dependent methods. Over the past decade, culture-independent methods have been applied to studies of human gut bacterial diversity where the majority of the bacteria are not culturable. These methods are helping scientists to better understand the role of human intestinal microbiota on human health and illness with a broader and less biased viewThe aim of our study is to characterize the fecal microbial community in patients with alcoholic liver cirrhosis, HBV-related liver cirrhosis and healthy controls. The fecal microbial community was analyzed by454pyrosequencing of the16S ribosomal RNA (16S rRNA) V3region followed by functional gene array GeoChip. Community-wide changes of fecal microbiota in liver cirrhosis were observed compared with healthy controls. The proportion of phylum Bacteroidetes was significantly reduced (p=0.008), while Proteobacteria and Fusobacteria were highly enriched in the cirrhosis group (p=0.001and0.002, respectively). Enterobacteriaceae (p=0.001), Veillonellaceae (p=0.046), and Streptococcaceae (p=0.001) were prevalent in cirrhotic patients at the family level. A positive correlation was observed between Child-Pugh (CP) score and Streptococcaceae (R=0.386, p=0.02). Lachnospiraceae significantly decreased in the cirrhotics (p=0.004), and correlated negatively with CP score (R=-0.49, p=0.002). By Partial Least Square Discriminate Analysis (PLS-DA), we identified149operational taxonomic units (OTUs) as key phylotypes that responded to cirrhosis, and most of them were Lachnospiraceae (65OTUs), Streptococcaceae (23OTUs), and Veillonellaceae (21OTUs).GeoChip data showed that the microbial community functional composition and structure were dramatically altered in the alcoholic cirrhosis and HBV-related cirrhosis. A variety of functional genes relevant to nutrient metabolism were significantly under-represented in cirrhotic patients than in controls, including genes for amino acid metabolism, lipid metabolism, cofactor biosynthesis and isoprenoid biosynthesis. Significant correlations were observed between functional genes and Child-Pugh score, such as aspartate-ammonia ligase gene,transaldolase gene, adenylosuccinate synthetase gene and IMP dehydrogenase gene.The454data showed significant increase of Prevotellaceae in alcoholic cirrhosis patients than in HBV-related cirrhosis patients. Various microbial functional genes involved in organic remediation, stress response, antibiotic resistance and virulence were highly enriched in the alcoholic cirrhosis group compared with control group and HBV-related cirrhosis group. Long term consumption of alcohol may stimulate microbial activities in stress response, organic remediation, antibiotic resistance and virulence.Conclusion:Fecal microbial communities are distinct in cirrhotic patients compared with healthy individuals. The prevalence of potentially pathogenic bacteria, such as Enterobacteriaceae and Streptococcaceae, with the reduction of beneficial populations such as Lachnospiraceae in cirrhotics may affect prognosis. Also, cirrhosis may have distinct influences on metaobolic potential of fecal microbial communities, which may have an effect in the progression of malnutrition.更多还原