沙门菌外膜囊泡对小鼠髓源树突状细胞的免疫调控作用  

Immune Modulatory Effect of Outer Membrane Vesicles Derived from Salmonella on Mouse Bone Marrow-Derived Dendritic Cells

作　　者：韦莉[1,2] 陈光璋 孙潮 张涛 管俊昌[1,2] 金齐力 WEI Li;CHEN Guang-zhang;SUN Chao;ZHANG Tao;GUAN Jun-chang;JIN Qi-li(Department of Microbiology and Parasitology,Bengbu Medical College,Bengbu 233030,China;Anhui Provincial Key Laboratory of Infection and Immunity at Bengbu Medical College,Bengbu 233030,China;Department of Clinical Laboratory,the Second Affiliated Hospital of Bengbu Medical College,Bengbu 233040,China)

机构地区：[1]蚌埠医学院病原生物学教研室,蚌埠233030 [2]感染与免疫安徽省重点实验室,蚌埠233030 [3]蚌埠医学院第二附属医院检验科,蚌埠233040

出　　处：《四川大学学报（医学版）》2021年第6期948-953,共6页Journal of Sichuan University(Medical Sciences)

基　　金：安徽高校科研创新平台团队项目(No.2016-40);安徽省高校优秀青年人才支持计划项目(No.gxyq2018037)资助。

摘　　要：目的研究鼠伤寒沙门菌(Salmonella typhimurium,ST)外膜囊泡(outer membrane vesicles,OMVs)对树突状细胞(dendritic cells,DC)超微结构及免疫功能的影响。方法无菌取小鼠骨髓细胞,体外经重组鼠源粒细胞/巨噬细胞集落刺激因子(recombinant mouse granulocyte-macrophage colony-stimulating factor,rm GM-CSF)和重组鼠源白细胞介素-4(recombinant mouse interleukin-4,rm IL-4)联合诱导扩增出DC,倒置相差显微镜下观察细胞形态,流式细胞术鉴定表型;超速离心分离获取ST-OMVs,CCK-8法测其对DC存活情况的影响,确定OMVs刺激浓度;透射电镜观察细胞负载OMVs后超微结构特征;流式细胞术检测DC细胞因子分泌、表面分子表达、吞噬能力。结果体外诱导和扩增的DC具有典型的细胞形态,纯度达85%以上,透射电镜可见DC表面有大量丝状伪足;OMVs负载后,可见DC表面树突状结构减少,胞浆内有大量囊泡状结构,线粒体增多,肿胀以及细胞凋亡;OMVs 5μg/mL和10μg/mL处理后,DC吞噬能力下降(P<0.05),成熟表型特征性分子表达上调(P<0.05),肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-1β(interleukin-1β,IL-1β)表达升高(P<0.05)。结论 ST-OMVs可诱导DC分泌TNF-α和IL-1β,具有促进DC成熟和抗原提呈的能力。Objective To study the effect of outer membrane vesicles(OMVs) derived from Salmonella typhimurium(ST) on the ultrastructural features and immune function of dendritic cells(DC).Methods Mice bone marrow cells were collected aseptically,and myeloid DC were generated by the combined induction and amplification with recombinant mouse granulocyte-macrophage colony-stimulating factor(GM-CSF) and recombinant mouse interleukin-4(rm IL-4).Cell morphology was observed under inverted phase contrast microscope and the phenotype was identified with flow cytometry.ST-OMVs were isolated through ultracentrifugation.The survival rate of DC was assessed with CCK-8 assay,and the stimulus concentration of OMVs was henceforth determined.The ultrastructural characteristics of DC loaded with OMVs were observed with transmission electron microscopy.The cytokine secretion,surface molecule expression and phagocytic capacity of DC were examined with flow cytometry.Results The DC induced and amplified in vitro displayed typical DC phenotype in morphological analysis and the purity of DC exceeded85%.Transmission electron microscopy showed that there were large numbers of protrusions on the cell surface.After stimulation with ST-OMVs,it was observed that the dendritic structures on the surface of DC were reduced and a large number of phagolysosomes were found in the cytoplasm.In addition,increased numbers of mitochondria,swelling and typical apoptosis were observed.After treatment with ST-OMVs at 5 μg/mL and 10 μg/mL,the secretion of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) of DC increased significantly(P<0.05).Furthermore,the immature DC could differentiate into mature DCs after stimulation with ST-OMVs,which were characterized by a decrease in phagocytic capacity(P<0.05) and an upregulation of phenotypic markers(P<0.05).Conclusion ST-OMVs can stimulate DC to produce TNF-α and IL-1β and promote DC maturation and antigen presentation.

关 键 词：鼠伤寒沙门菌 外膜囊泡 树突状细胞 

分 类 号：R392[医药卫生—免疫学]