乳头切开术后肠胆反流引起微生态改变致复发性胆管结石的机制研究

内镜下乳头切开（EST）术后复发性胆管结石发生率高且具体机制不明。以往研究显示细菌在胆管结石形成中发挥重要作用，课题组前期研究表明胆管结石与非胆管结石患者肠道菌群及胆汁成分均存在明显差异。本研究拟通过构建肠胆反流模型（EST）及抗肠胆反流模型(内镜下乳头成型术，EEPP)，验证EST术后肠胆反流引起肠道菌群改变、胆汁微环境变化是复发性胆管结石的启动机制，并初步探讨二者的相互作用规律。经由干预微生物群落（益生菌、抗生素、致病菌）和核素示踪大肠杆菌等手段，进一步验证肠道菌群反流入胆管并利用生物膜定植是胆石形成的关键步骤。此外，采用构建的基因缺陷和过表达菌株，通过细菌生长曲线、蛋白及基因表达水平检测、生物膜形成试验（体外生成测定、荧光检测和直接观察、小鼠FISH检测）、猪模型胆管结石形成实验等方法，探索细菌生物膜、β-葡萄糖醛酸酶、磷脂酶A1的调节机制及其在胆管结石形成中的作用。

Endoscopic Sphincterotomy（EST）based stone extractions was the predominant therapy for choledocholithiasis. However, the mechanism of high recurrence rate of choledocholithiasis was still not known. Researchers had demonstrated that bacteria played an important role in common bile duct stone formation. Our previous work showed that intestinal microecology and biliary microenvironment was different between choledocholithiasis and non-choledocholithiasis patients. In this study, we attempted to establish two type of models, one was EST as duodenal-biliary reflux model, and the other is Endoscopic Endoclip Papilloplasty as anti-duodenal-biliary reflux model. Based on these two models, changes of gut flora, bile acids concentration and bile contents were investigated. Role of duodenal refluxed and bile duct colonizing bacteria in bile duct stone formation was tested, by adding probiotics, antibiotic or pathogens to intervene gut flora, or using radionuclide labelling E. coli. Moreover, bacteria growth-curve, protein and gene expression level, biofilm formation test (ex vivo formation test, florescence detection and FISH test in mice), bile duct stone formation in pig models were conducted in gene knocking-out or over-expression bacteria. Thus, the regulating mechanisms and their roles in bile stone formation of bacteria biofilm, β-glucuronidase and phospholipase A1 were discovered.