南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (05): 727-732.doi: 10.12122/j.issn.1673-4254.2020.05.19

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弓形虫感染导致DC2.4细胞外泌体特征差异的研究

李东亮，杨淑君，彭鸿娟   

  • 出版日期:2020-05-20 发布日期:2020-05-20
  • 基金资助:

Characteristics of exosomes secreted by Toxoplasma gondii-infected mouse dendritic DC2.4 cells

  

  • Online:2020-05-20 Published:2020-05-20

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摘要： 目的 分析小鼠树突状细胞（DC2.4）感染弓形虫后所分泌的外泌体及其富集的小RNA与piRNA的变化，为外泌体在弓形虫-宿主互作中的作用提供必要的信息。方法 DC2.4细胞分为2组（未感染组与感染组），用含无外泌体血清的培养基培养。待细胞长满，感染组用弓形虫RH株速殖子感染28 h，未感染组则培养28 h。收集两组细胞的培养上请，采用超速离心法分别提取外泌体，通过透射电镜检测外泌体形态，并用纳米颗粒追踪仪检测外泌体的粒径大小以及颗粒密度。对外泌体进行小RNA高通量测序分析，用生物信息学方法分析感染组与对照组差异显著的piRNA，对并其靶基因及功能进行预测。结果 对弓形虫感染与未感染的DC2.4细胞分泌的外泌体进行形态学鉴定以及粒径大小和密度的比较，发现弓形虫感染会导致DC2.4分泌的外泌体密度增加；而通过外泌体小RNA测序，发现弓形虫感染会导致外泌体中 miRNA及piRNA数目增加，其中感染后显著升高的piRNA包括piR-mmu-159、piR-mmu-1526、piR-mmu-9082、piR-mmu-17405、和piR-mmu-25576。对上述piRNA靶基因的预测和KEGG分析，显示这些靶基因显著富集到MAPK、Ras、cAMP、actin细胞骨架调节、粘着斑形成、轴突导向等信号通路上。结论 弓形虫感染导致DC2.4细胞分泌的外泌体粒径尺寸峰值增大和密度增加，miRNA及piRNA数目增加，提示外泌体在弓形虫-宿主细胞互作中起着重要作用。

Abstract: Objective To investigate the changes in the exosomes secreted by mouse dendritic cell line DC2.4 after infection with Toxoplasma gondii and to analyze the possible regulatory mechanisms underlying such changes. Methods The exosomes were extracted by ultracentrifugation from DC2.4 cells at 28 h after infection with Toxoplasma gondii. The morphology of the exosomes was examined with transmission electron microscopy, and the exosome size and density were determined using a nanoparticle tracker. High-throughput sequencing was carried out to identify the differentially expressed small RNAs in the exosomes derived from the infected cells. Results T. gondii infection resulted in a significantly increased density of exosomes secreted by DC2.4 cells. Small RNA sequencing revealed that Toxoplasma infection caused an increase in the number of miRNAs and piRNAs in the exosomes. The significantly up-regulated piRNAs after the infection included piR-mmu-159, piRmmu-1526, piR-mmu-9082, piR-mmu-17405, and piR-mmu-25576. Conclusion Toxoplasma infection causes accumulation and enrichment of exosomes secreted by DC2.4 cells with increased miRNAs and piRNAs in the exosomes.