Immortalization Of The Target Cell Of SFTSV And RNA Sequencing Analysis

Objective:To isolate,purify and culture fibroblastic reticular cells(FRCs)of mouse spleen,to develop a reliable and robust method to immortalize primary mouse FRCs,to filter stable FRCs cell lines,to learn the susceptibility of FRCs to SFTSV and explore the driver genes and associated pathways of this cells after infection.Method:After purifying FRCs by fluorescence activated cell sorting(FACS)from auto MACS-enriched stroma cells of mouse spleen,we infected this cells by simian virus 40 large T antigen and SFTSV in vitro,screened the FRCs clones with puromycin,compared primary and immortalized FRCs by RNA sequencing(RNA-seq)and Confocal Microscopy technology,isolated differentially expressed genes(DEGs)between normal and infected FRCs by RNA sequencing(RNA-seq)technology,assigned those DEGs in signal transductions,metabolic and biosynthesis pathways with Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.Result:We succeed in culturing purified FRCs(purity: 99%),isolated two stable FRCs clones: Clone01,Clone02,both of which survived for more than 50 passages.Clone01 lost the typical FRCs-like morphology,the rate of expansion of Clone01 is quite different with that of primary FRCs and Clone02.The gene expression pattern of Clone02 is closer to primary FRCs than Clone01,find 5914 DEGs among which 2815 upgraded and 3099 downgraded and Significant changed 33 Go terms,20 pathways.We show top ten DEGS,filtered GO terms such as plasma membrane,Golgi apparatus,endoplasmic reticulum,immune system process,regulation of transcription from RNA polymerase II promoter,virion,virion part and so on,enriched RNA polymerase,Ribosome,Ribosome biogenesis in eukaryotes,m RNA surveillance,RNA transport Lysine degradation,Peroxisome pathways,Wnt?TNF?Notch?MAPK signal pathways etc.Conclusion:The stable FRCs clone Clone02 has FRCs-like morphology and express key FRCs surface markers podoplanin(GP38 or PDPN)and do not express endothelial cells markers CD31 and leukocyte common antigen CD45.Additionally,it was confirmed to be of mouse origin by sequencing.The RNA expression profiles identified by RNA-seq are also characteristic of FRCs,FRCs can be stably cultured for a short period of time and infected by SFTSV in vitro.Results from GO analysis are consistent with that of KEGG pathways,they will help characterize the detailed mechanisms of infection.