单细胞测序技术解析重要肠道菌对肠道上皮再生与分化的影响

Gastrointestinal tract is an important digestive and immune organ, hosting a large number of microorganisms. Intestinal epithelial cells respond to microbes and function as a barrier against pathogens, as well as to coordinate immune responses. The disturbance of the host-microbial interaction in the intestines can induce a variety of inflammatory diseases and metabolic syndrome. Intestinal epithelium maintains intestinal permeability and homeostasis through the continuous proliferation and differentiation of stem cells, which requires multiple regulatory mechanisms, including nutrient factors, signal factors and transcription factors. Importantly, responses of epithelial cells to bacteria have a key role in maintaining gut homeostasis but there is lack of comprehensive study on this topic. In this study, we will first establish intestinal organoid culture system and co-culturing intestinal organoids with A.muciniphila and F.nucleatum. The organoids system can effectively replicate the microenvironment of originating organ and manipulate many affecting factors to maintain the functionality, molecular and cellular heterogeneity. Next, single-cell transcriptome profiling will use to construct landscape of intestinal epithelial proliferation and differentiation, in germ-free and bacterial co-culturing intestinal organoids. As a result, we will identify the essential role of bacteria in intestinal epithelial development, further investigate responses of intestinal epithelial cells to A.muciniphila and F.nucleatum, and elucidate the dynamic expression profiles as well as regulatory functions in intestinal epithelial proliferation and differentiation. We hope to provide a systemic understanding of intestinal epithelial proliferation, a new theoretical basis and technological guidance for maintenance of intestinal homeostasis.

肠道是重要的消化和免疫器官，与肠道微生物组有复杂的相互作用，共同影响宿主健康，与微生物共生关系的扰动则会诱发多种炎症疾病及代谢综合症。肠道上皮通过干细胞的不断增殖分化来维持肠道通透性、保持稳态，其再生和分化需要多种营养因子、信号因子及转录因子的调控。细菌在其中起到非常重要的作用但至今尚未有精细、全面的阐述。伴随多种技术的发展，目前有机会进行深入的研究。本课题将建立无菌、与益生菌A.muciniphila和致病菌F.nucleatum共培养的小肠类器官系统，控制多种变量并有效模拟机体微环境，用于研究单菌如何影响肠道的分化。在此基础上，通过单细胞测序技术绘制无菌及有菌条件下小肠类器官再生和分化的单细胞图谱，阐述细菌对肠上皮谱系分化和动态表达的影响。发掘调控肠道分化的关键性细菌分子、潜在信号通路和调控网络，探索关键性分子调控肠道分化的机制，为临床治疗中维持肠道稳态提供重要的理论依据和技术指导。

肠道是重要的消化和免疫器官，其上寄居着大量微生物,肠道细菌与肠道上皮的互作可以调控宿主的生长发育、免疫防御、物质代谢、衰老及内分泌等多种重要生物过程。肠道上皮的再生和分化受到营养因子、信号因子及转录因子的调控。这些通路及因子的异常会导致肥胖、营养不良以及肠道炎性疾病，细菌在其中起到非常重要的作用。本项目建立了无菌及分别与细菌Fusobacterium nucleatum和Akkermansia muciniphila共培养共培养的小肠类器官系统；并建立单细胞分析流程，无菌及有菌条件下小肠类器官再生和分化的单细胞图谱；以无菌组为基线，解析益生菌及致病菌对肠道上皮分化和动态表达的影响，获得调控肠上皮分化过程中的多个关键分子及其调控的信号通路；并发现其中一个RNA结合蛋白PCBP1可能帮助有益菌Akkermansia muciniphila增加小肠上皮细胞的分化。并通过体外实验，进一步证明PCBP1通过影响mRNA核质穿梭调控干细胞分化。