基于单细胞转录组学解析猪iPSCs分化为PAM的分子机制

猪肺泡巨噬细胞（PAM）是猪机体重要的免疫细胞，也是猪繁殖与呼吸综合征病毒(PRRSV)侵染的主要靶细胞，因此是研究PRRSV与宿主互作机制的理想模型。然而由于对PAM发育过程及其生物学特性不清楚，导致缺乏能大量扩增且功能活性良好，能用于抗PRRSV感染新靶点筛选的细胞株，阻碍了PRRSV感染与致病机制的深入研究。申请人前期已建立了稳定的猪iPSCs系，具有无限增殖能力和分化为PAM的潜能。本项目拟以PAM的标记/PRRSV受体—CD163作为报告基因，建立猪iPSCs诱导分化为iPAM的体外培养体系，再现从干细胞到PAM的生物学过程；用单细胞转录组测序技术系统鉴定决定PAM发育分化及生物学特性的关键因子；进行PRRSV感染，测序、比较病毒感染情况，挖掘抗PRRSV感染新靶点，研究其生物学功能、评价PRRSV的感染和增殖情况。为抗病育种、疫苗生产提供坚实的理论基础和稳定的细胞模型。

Porcine alveolar macrophage (PAM) is the main target cell of PRRS virus (PRRSV) infection and an ideal model for studying the mechanism of interaction between PRRSV and host. However, due to the unclear development process and biological characteristics of PAM especially for the key genes determining the function of PAM, so there is a lack of cell line that can be extensively amplified and used for screening new PRRSV targets, which hindering the in-depth study of PRRSV infection and pathogenesis. Stable porcine iPSCs lines have been established in our laboratory, which has the capacity of unlimited proliferation and the potential to differentiate into many cell types of the body including PAM. This project intends to establish an in vitro culture system of porcine iPSCs induced differentiation into PAM by PAM marker and PRRSV receptor -CD163 as the reporter gene, and generate the functional PAM and reproduce the biological process from pluripotent stem cells to PAM. Single cell RNA-seq will be used to identify the key factors that determine the development, differentiation and biological characteristics of PAM. The cells infected was sequenced and compared with non-infected to find new anti-PRRSV targets, study their biological functions, and evaluate the infection and proliferation of PRRSV, so as to provide a solid theoretical basis and a stable cell model for animal disease resistance breeding and vaccine production.