基于CD169表达水平的小鼠脾红髓巨噬细胞分型研究

Definition of macrophage subtypes based on the expression of CD169 in murine splenic red pulp macrophages

目的·基于小鼠脾红髓巨噬细胞唾液酸黏附蛋白(sialoadhesin,Sn)即CD169分子的表达水平,研究CD169+和CD169-红髓巨噬细胞亚群的基因表达谱差异。方法·采用流式细胞术和免疫荧光法检测C57BL/6J野生型(WT)小鼠脾红髓巨噬细胞中CD169的表达,以CD169敲除(CD169 KO)小鼠为阴性对照。富集F4/80+的脾红髓巨噬细胞并分选出CD169+和CD169-2个亚群,进行RNA测序。利用DESeq2软件以P<0.05且|log2FC|≥1为条件筛选出差异表达基因,通过京都基因与基因组百科全书(Kyoto Encyclopedia of Genes,KEGG)功能分析,将差异基因按照参与的通路或功能进行分类。通过实时荧光定量PCR(quantitative realtime PCR,qPCR)验证部分差异基因。结果·流式细胞术和免疫荧光法证实部分红髓巨噬细胞表达CD169。CD169+和CD169-亚群拥有485个差异表达基因。一些与介导炎症相关的基因在CD169-亚群中高表达。结论·CD169+和CD169-红髓巨噬细胞有不同的转录谱,CD169-红髓巨噬细胞亚群具有更多M1型巨噬细胞的特征。

Objective·To investigate the gene expression profiles of CD169+and CD169-red pulp macrophages based on the expression of CD169 in murine splenic red pulp macrophages.Methods·The expression of CD169 in splenic red pulp macrophages in C57BL/6 wild-type(WT)mice was analyzed by flow cytometry and immunofluorescence.CD169 knockout(KO)mice were used as negative control.F4/80+splenic red pulp macrophages were enriched and separated into CD169+and CD169-subtypes.RNA-sequencing was performed on the two subtypes.DESeq2 was used to analyze differentially expressed genes at P<0.05&|log2FC|≥1.Kyoto Encyclopedia of Genes(KEGG)enrichment analysis was used to classify the differentially expressed genes according to the pathways involved or functions they performed,and some differentially expressed genes were verified by quantitative real-time PCR(qPCR).Results·Expression of CD169 in some red pulp macrophages was confirmed by flow cytometry and immunofluorescence.There were 485 differentially expressed genes in CD169+and CD169-subtypes.Some differentially expressed genes related to inflammation were highly expressed in the CD169-subtype.Conclusion·The CD169+and CD169-red pulp macrophages have different transcriptional profiles,and CD169-red pulp macrophages have more features of M1 macrophages.