SFTS病毒感染脾脏网状成纤维细胞系的建立

Immortalization of spleen fibroblastic syndrome virus

目的分离、纯化、培养和永生化小鼠脾脏原代网状成纤维细胞（fibroblastic reticular cells, FRCs），筛选和鉴定永生化的FRCs克隆，证明永生化FRCs可用于SFTS病毒（SFTSV）体外感染研究。方法使用磁珠和流式细胞分选等技术分离、纯化和培养小鼠脾脏FRCs；使用SV40永生化FRCs，筛选单克隆并培养50代以上，使用转录组测序（RNA Sequencing, RNA-seq）和激光共聚焦技术对原代和永生化的FRCs进行比较和鉴定；用SFTSV体外感染的永生化的FRCs。结果成功得到4个永生化的FRCs克隆；鉴定结果显示Clone02符合FRCs表型，且纯度均达到了99%；转录组测序相关性分析显示Clone02细胞株基因表达与原代FRCs接近；基因表达分析和激光共聚焦实验证实Clone02的表型和原代FRCs相同；SFTSV在MOI值0.5的条件下即可成功感染Clone02细胞。 结论永生化的FRCs Clone02细胞保持了原代细胞的形态学、免疫学特征，与原代FRCs基因表达谱接近，可被SFTSV体外感染，是一个新的有潜力的SFTSV体外研究的细胞平台。

ObjectiveTo isolate, purify and culture fibroblastic reticular cells （FRCs） of mouse in spleen, to develop a reliable and robust method to immortalize primary mouse FRCs, to filter stable FRCs cell lines, to prove that the clones can be infected by SFTSV in vitro.MethodsAfter purifying FRCs by fluorescence activated cell sorting （FACS） from autoMACS-enriched stroma cells of mouse spleen, we infected FRCs by simian virus 40 large T antigen in vitro, screened the FRCs clones with puromycin, compared primary and immortalized FRCs by RNA sequencing（RNA-seq） technology, infected the clones with severe fever with thrombocytopenia syndrome virus （SFTSV） in vitro.ResultsWe succeed in culturing purified FRCs from spleen, isolated four stable FRCs clones, two of which have a purity of 99%, survived for more than 50 passages, express the key FRCs marker podoplanin and do not express CD31 and CD45. Clone 01 lost the typical FRCs-like morphology, the rate of expansion of which is quite different from that of primary FRCs and Clone 02. Clone 02 can be infected with SFTSV, which has the same gene expression pattern and immunophenotype with primary FRCs.ConclusionsThe stable FRCs clone Clone 02 has FRCs-like morphology and express key FRCs surface markers podoplanin （GP38 or PDPN） and do not express endothelial cell markers CD31 and leukocyte common antigen CD45. The RNA expression profiles identified by RNA-seq are also characteristic of FRCs. Infected with SFTSV in vitro, Clone 02 will be a new platform to study SFTSV.