羊口疮病毒对树突状细胞成熟和功能的影响及转录组学分析

Effect of Orf Virus on the Maturation and Function of Dendritic Cells and Transcriptomics Analysis

羊口疮病毒(Orf virus,ORFV)感染机体后能够引起强烈的免疫应答反应。为了探究ORFV对小鼠骨髓来源树突状细胞(Bone marrow-derived dendritic cells,BMDC)成熟及未成熟的CD4^+T细胞(Na?ve CD4^+T细胞)极化作用,并且寻找ORFV感染BMDC后与免疫相关的差异表达基因。本研究用不同感染复数(Multiplicity of infection,MOIs)的ORFV感染BMDC,检测ORFV的感染是否能够促进BMDC成熟,即通过测定其表面共刺激分子表达情况、细胞因子的分泌情况以及细胞的吞噬功能判断BMDC是否能够成熟。使用流式方法检测该细胞对Na?ve CD4^+T细胞刺激能力的影响,并对1 MOI ORFV作用后的BMDC进行高通量测序。结果表明1 MOI ORFV能够促进BMDC的成熟,并且能诱导Na?ve CD4^+T细胞向Th1细胞分化。高通量测序共检测到有8241个差异表达基因,其中上调基因4 205个,下调基因4036个。这些差异表达基因主要参与抗原递呈与加工过程、T细胞受体信号通路、T细胞激活与细胞因子分泌途径以及细胞凋亡信号通路中等。荧光定量逆转录-聚合酶链反应(Reverse transcription-polymerase chain reaction,RT-PCR)对部分差异表达基因进行验证,结果与RNA-seq测序结果一致。本研究为进一步阐明ORFV与免疫细胞的相互作用机制奠定基础。

Orf virus(ORFV)can cause a strong immune response after infection. To investigate the maturation of mouse bone marrow-derived dendritic cells(BMDCs)and the polarization of na?ve cluster of differentiation(CD)4^+T cells by ORFV and find immunologically differentially expressed genes(DEGs) after ORFV infection of BMDCs,BMDCs were infected by ORFV with different multiplicity of infection(MOI)values.Whether the cell could mature after infection was tested. The expression of surface co-stimulatory molecules and the secretion of cytokines were determined;the phagocytotic function of BMDCs was also detected. The effect of the cells on the stimulatory ability of na?ve CD4^+T cells was detected. High-throughput sequencing of BMDCs infected by 1 MOI was done. We found that ORFV of 1 MOI could promote the maturation and differentiation of na?ve CD4^+T cells into Th1 cells. A total of 8241 DEGs were detected by RNA-seq sequencing(4205 up-regulated genes and 4036 down-regulated genes). These genes showed different antigen presentation/processing,T-cell receptor signaling pathway,T-cell activation and cytokine secretion pathways,and apoptotic signaling pathways. Partially DEGs were verified by fluorescence quantitative reverse transcription-polymerase chain reaction,and the results were consistent with the result of transcriptome sequencing. These data lay the foundation for further elucidation of the mechanism of ORFV and immune cells.