肠道微生态失衡与肝衰竭发病机制的研究

Liver failure is a complicated clinical syndrome, also easily caused intercurrent infection, with high mortality. More and more evidence proved that the intestinal microecology plays an important role in the development of hepatic failure. After being hit, function of intestinal barrier is impaired, and intestinal bacteria and endotoxin entered into the portal system. The macrophages in the liver (kupffer cells) are activatied by the endotoxin, which released a series of inflammatory factor. Various cytokines inflammation interacted and a complicated network structure was emerged, which destroyed the intestinal mucosa and remote organ. The traditional microbial research, such as culture technology or the method of molecular biology are low throughput, time-consuming and low accuracy, which can't detect microbiome in trace level. The second generation sequencing technology, with high accuracy, high throughput, high sensitivity, promote the development of intestinal microecology. In this study, we aim to collect 100 fecal samples from healthy people(50) and liver cirrhosis patients(50), extract genomic DNA of feces flora, amplify 16 s rDNA, and use Illumina-based sequencing to find out the detail of micro ecological unbalance and expect to find the marker of the microflora. Then we take 20 healthy volunteers and 20 patients, using the metagenomic sequencing to find out the change of functional gene, intends to find the functional genes maker of hepatic failure disease. We try to clarify the relationship between intestinal flora and intestinal tract, reveal the pathogenesis of liver failure from the view of micro-ecology, and provide the theoretical basis to the early warning of liver failure and development of intestinal probiotics.

肝衰竭是临床常见症候群，极易并发感染，病死率极高。肠道微生态在肝衰竭的发生发展中起重要作用。肠道遭受打击后肠屏障功能受损，细菌和内毒素易位，肝内巨噬细胞被激活，释放的各种细胞因子炎症介质相互作用形成复杂的网络，从而进一步损害肠道黏膜及远隔器官。传统培养以及分子生物学的方法技术通量低、耗时、准确性低，无法检测痕迹量微生物，以第二代测序技术作为研究手段的宏基因组学技术以其高准确性，高通量，高灵敏度，和低成本等突出优势迅速推动了微生态学的发展。本课题计划收集50例健康人及50例肝衰竭患者的粪便样本，对患者肠道微生物进行16S V4区扩增、测序及生物信息学分析，并对其中的健康志愿者20例、乙肝肝衰竭患者20例进行粪便菌群宏基因组研究，期望找到和肝衰竭病情转归的标志菌群及其功能基因，阐明菌群与肠道相互作用关系，从微生态的角度揭示肝衰竭的发病机制，为肝衰竭的早期预警及肠道微生态制剂的研发提供理论基础。

肝衰竭是临床常见症候群，极易并发感染，病死率极高。肠道微生态在肝衰竭的发生发展中起重要作用。肠道遭受打击后肠屏障功能受损，细菌和内毒素易位，肝内巨噬细胞被激活，释放的各种细胞因子炎症介质相互作用形成复杂的网络，从而进一步损害肠道黏膜及远隔器官。传统培养以及分子生物学的方法技术通量低、耗时、准确性低，无法检测痕迹量微生物，以第二代测序技术作为研究手段的宏基因组学技术以其高准确性，高通量，高灵敏度，和低成本等突出优势迅速推动了微生态学的发展。本课题收集28例健康人及28例慢性肝衰竭患者的粪便样本，进行宏基因组小片段建库、测序及生物信息学分析。宏基因组测序共产生了252G的测序数据，平均每个样品产生4.49 GB的数据，建立了第一个慢性肝衰竭患者的基因集，共包含596,075个基因，平均基因长度为802 bp，与之前肝硬化的基因集比较，建立了新的肝病基因集，共包含2,909,229个基因，其中7.6%即220,759个为肝衰竭患者所独有的基因。与健康对照相比，慢性肝衰竭患者的肠道微生物存在显著的差异，研究共发现了67303个基因标记物，其中35562个基因标记物在病例组中富集，31741个在健康组中富集。通过聚类，共发现了27个与疾病相关MGS，其中12个在病人组中富集，15个健康人中富集，基于这些组群，建立了肝衰竭诊断模型，AUC值为0.966（95% CI：0.919~1.000）。功能分析发现，差异基因注释到KEGG的有1799个，其中健康人主要集中在细胞运动，信号转导和碳水化合物代谢，病人主要集中在基因转录，翻译和复制，Module分析发现慢性肝衰竭患者肠道菌群显著富集基因还涉及磷酸多糖合成和转运（与生物膜形成相关），γ-氨基丁酸（GABA）生物合成（肝硬化肠道菌群中也有类似发现）以及氨基酸运输相关。本研究揭示了慢性肝衰竭患者肠道菌群结构和功能基因的显著变化，并发现了这一变化与疾病之间的显著相关性，为医疗诊断提供了一种新的检测方法，同时对疾病的微生态干预治疗提供了理论基础。