特征肠道菌群影响非酒精性脂肪性肝炎的作用机制及其在临床诊断中的应用

Non-alcoholic steatohepatitis (NASH) can progress to sever liver diseases, including liver cirrhosis and hepatocellular carcinoma, and is strongly associated with gut microbiota. Using 16S rRNA gene V3-V4 amplicon metagenomics, we found there were significantly differences in gut bacterial structures between NASH and the control, and identified host phenotype-specific bacteria (HPSB) as biomarkers. However, whether and how these bacteria affect the development and progression of NASH and what their indication is in new clinical samples are unknown, since the results are based on sequencing data. Therefore, this proposal plans to first isolate diverse bacteria from the sequenced fecal samples, and screen strains that match the biomarkers in sequencing results as potential HPSB. By means of shotgun metagenomics, Western-blot, qPCR, immunohistochemical staining, etc, we will further verify the effects and reveal the mechanisms of HPSB in NASH in aspects of gut microbial (bacterial, fungal, viral) community structures, short-chain fatty acids, gut permeability, endotoxin LPS, and liver inflammatory factors. Finally, we will evaluate the clinical value of HPSB by designing highly specific qPCR primers, measure HPSB abundances, and establish a potential diagnostic index using machine learning methods. Results of this proposal will not only reveal the mechanisms of HPSB in NASH, but also provide a potential rapid method for the clinical diagnosis.

非酒精性脂肪性肝炎（NASH）可发展为肝硬化、肝癌等严重肝脏疾病，且与肠道微生物有着密切的关系。本实验室通过高通量测序分析发现，NASH患者和健康对照的肠道细菌群落结构存在显著差异，并鉴定到可作为生物标志物的特征菌群，但这些特征菌群是否以及如何影响NASH的发生发展及其在新的临床样本中的指示作用急需实验数据支持。本课题拟结合最新肠道细菌纯培养技术，从已测序粪便中分离鉴定出与测序结果相吻合的潜在特征菌株；在细胞和动物模型水平，采用宏基因组学、Western-blot、qPCR、免疫组化等技术，从肠道微生物落结构、肠粘膜通透性、血清内毒素水平、肝细胞炎症反应等方面验证和揭示特征菌群影响NASH的作用机制；进而针对特征菌群设计特异性引物，在临床样本中检验特征菌群的丰度与特异性，提出诊断候选指标及其基线值。研究结果不仅能揭示特征肠道菌群影响NASH的作用机制，也将为NASH的快速诊断提供新思路。

近年来，非酒精性脂肪性肝病（NAFLD）发病率不断上升，已成为世界上最常见的肝脏疾病。自“肠肝轴”概念提出以来，肠道与肝脏疾病的关系，尤其是肠道微生物与非酒精性脂肪性肝病（NAFLD）的相关性、发病机理和临床治疗等方面的研究越来越受到重视。本课题通过对NAFLD患者肠道菌群进行分离培养，获得多株与NAFLD相关细菌。动物实验发现Escherichia fergusonii可有效缓解HFD小鼠肝脏脂肪堆积，减轻肝脏脂肪变，改善HFD小鼠的肝脏炎症，确了Escherichia fergusonii可参与对NAFLD发生发展调控，揭示了补充肠道特征菌Escherichia fergusonii在NAFLD中发挥功能与作用机制，为预防与治疗疾病提供了潜在的新策略。通过数据库多中心公开数据的下载分析，构建了基于肠道细菌的NAFLD分类诊断模型，为临床实践中NFALD的无创诊断提供了新方向，将有助于开发快捷、无创的诊断方法。另外进行了扩展性研究阐述了宿主基因STING缺失伴随肠道菌群丰度的改变，改善了HFD诱导的NAFLD肝脏炎症和脂肪变，为阐述疾病发生发展中宿主基因和肠道微生物间的关系提供了新的思考。