奇异变形杆菌源外膜囊泡诱导小鼠巨噬细胞向M1型和M2b型极化

Polarization of mouse macrophages towards M1 and M2b types induced by outer membrane vesicles derived from Proteus mirabilis

为探究条件致病菌奇异变形杆菌(Proteus mirabilis)分泌的细菌外膜囊泡(outer membrane vesicles,OMVs)是否具备诱导巨噬细胞极化以调控肿瘤微环境的功能,本研究以该OMVs刺激小鼠巨噬细胞RAW264.7,检测巨噬细胞极化相关表型。首先,利用透射电镜对分离提取的OMVs进行表征;其次,用PKH67荧光染料示踪OMVs,通过荧光显微镜观察RAW264.7细胞对OMVs的摄取情况;最后,利用总一氧化氮(NO)检测试剂盒和实时荧光定量PCR (RT-qPCR),分别对RAW264.7细胞分泌的总NO含量和RAW264.7细胞极化相关因子表达进行检测。结果表明,OMVs可被RAW264.7细胞摄取并诱导该细胞NO分泌增多,同时上调IL-10、IL-1β、TNF-α和IL-6的表达,即发生M1型和M2b型极化,从而激活巨噬细胞的抗肿瘤活性。上述结果表明奇异变形杆菌分泌的OMVs在该菌抗乳腺肿瘤生长和肺脏转移中发挥潜在作用。

To investigate whether bacterial outer membrane vesicles(OMVs)secreted by the conditionally pathogenic bacterium Proteus mirabilis have the function of inducing macrophage polarization to regulate the tumor microenvironment,the mouse macrophage RAW264.7 was stimulated with the OMVs to detect macrophage polarization-related phenotype.The OMVs were isolated,extracted and then characterized using transmission electron microscopy.The OMVs were traced with PKH67 fluorescent dye,and the uptake of OMVs by RAW264.7 cells was observed by using fluorescence microscopy.The total NO content secreted by RAW264.7 cells and the expression of polarization-associated factors in RAW264.7 cells were detected by using total nitric oxide(NO)detection kit and real-time fluorescence quantitative PCR(RT-qPCR),respectively.The results showed that OMVs could be taken up by RAW264.7 cells,induced an increase in NO secretion in these cells,and up-regulated the expression of IL-10,IL-1β,TNF-α,and IL-6,i.e.,the occurrence of M1-type and M2b-type polarizations,which activated anti-tumor activity of macrophages.The above results suggest a potential role of OMVs secreted by P.mirabilis in the bacterium's resistance to breast tumor growth and lung metastasis.