肠道共生梭菌对肠杯状细胞分化成熟及相关抗原通道形成的机制研究

Dysfunction of goblet cell (GC) was involved in the pathogenesis of inflammatory bowel disease. At previous study, we found that proportion and abundance of Clostridium leptum and Clostridium coccoides group were decreased in patients with IBD, concomitant with increase of Escherichia coli group, and tanscriptional factors involved in GC differentiation Hath1 and Klf4 decreased in patients with IBD, while no significant changes were observed in upper stream signals of Notch and Wnt. In our preliminary experiment study, we found that expression of KLF4 increased in HT-29 and Hep2 cell line stimulated with commensal Clostridium spp, isolated from healthy populations, and expression of KLF4 increased in intestinal mucosa of IL-10 deficient mice with commensal Clostridium spp. Therefore, we hypothesize that Clostridium spp. can induce the differentiation and maturation of GC and contribute to the formation of goblet cell associated antigen passages and maintenance the integrity of intestinal barrier. We are going to demonstrate that an established intestinal crypt cell three-dimensional culture system can be used for the study of GC differentiation, and confirm the commensal Clostridium spp. can induce GC differentiation and maturation from intestinal and contribute to the formation of goblet cell associated antigen passages, and impacts of commensals Clostridium on crypt cell differentiation and formation of goblet cell associated antigen passages in vivo. These will give insights into the pathogenesis of inflammatory bowel disease.

炎症性肠病患者黏膜杯状细胞(goblet cell, GC)功能存在异常。我们前期研究发现：①IBD患者粪便中柔嫩梭菌群和球形梭菌群减少伴大肠杆菌群丰度增加，②IBD患者黏膜组织中Notch\Wnt表达未见异常而Hath1、Klf4表达下降。此外，我们的预实验结果显示：①在健康人粪便中分离出多株共生梭菌，②共生梭菌能刺激HT-29、Hep2细胞株KLF4表达增加，③共生梭菌能刺激IL-10-/-肠炎小鼠结肠黏膜中KLF4表达增加。我们推测：共生梭菌能诱导GC分化及功能成熟并促进相关抗原通道形成维持黏膜屏障完整及黏膜免疫稳态。我们拟进一步研究：①构建肠上皮隐窝细胞培养体系，体外模拟GC分化过程，②体外验证共生梭菌对肠隐窝细胞分化及杯状细胞相关抗原通道形成的影响，③体内验证共生梭菌对肠上皮杯状细胞功能及黏膜免疫耐受的影响。明确共生梭菌在GC分化成熟中的作用，为IBD的发病机制提供新的理论认识。

本研究利用肠黏膜隐窝干细胞类器官模型研究肠上皮细胞及黏膜屏障功能。临床研究发现具有较高肠道菌群多样性、及其代谢产物短链脂肪酸（short chain fatty acids, SCFAs）的CD患者对药物应答率、维持缓解率较高；动物模型证实SCFAs对肠黏膜免疫系统有直接的、积极的调节作用；但临床应用研究表明SCFAs灌肠治疗并不能改善肠炎。我们推测肠道菌群共生梭菌能诱导GC分化及功能成熟并促进相关抗原通道形成维持黏膜屏障完整及黏膜免疫稳态。.1).构建肠隐窝细胞三维培养体系，建立、验证及检测肠类器官模型；.2).购买并鉴定KO IL10-/-免疫缺陷型IBD模型小鼠，比较及验证野生型小鼠与KO IL10-/-小鼠肠上皮细胞分化及增殖水平；结论：在没有炎症刺激的生理条件下，IL10基因缺陷不会影响ISC的增殖和分化。.3).基于小鼠肠类器官模型，体外检测Faecalibacterium prausnitzii菌株及菌体成分细胞实验效应，检测肠类器官增殖及分化相关形态及标记性宿主基因表达水平；结论：Faecalibacterium prausnitzii代谢产物可有效促进肠类器官增殖，并促进肠隐窝干细胞lgr5基因表达IBD患者肠道产丁酸盐细菌分度下降；.4).基于小鼠肠类器官模型，体外验证共生梭菌主要代谢产物短链脂肪酸的效应；结论：肠类器官模型对SCFAs的形态学应答存在浓度适应性；.5).构建IBD病例-对照队列，完成临床实验伦理注册，完整采集患者信息及生物样本，利用肠道菌群16sRNA测序技术和靶向代谢组学技术，分别检测肠道菌群和肠道单链脂肪酸水平。