天津医药

天津医药 ›› 2022, Vol. 50 ›› Issue (7): 678-685.doi: 10.11958/20220154

• 细胞与分子生物学 • 上一篇    下一篇

Mtb感染Ⅱ型肺泡上皮细胞来源外泌体通过miR-145对巨噬细胞极化的影响

李建军，吴素方，白丰玺   

  1. 河南省胸科医院结核二科（邮编450008）
  • 收稿日期:2022-01-26 修回日期:2022-03-18 出版日期:2022-07-15 发布日期:2022-07-15
  • 基金资助:
    2019年度河南省医学科技攻关计划联合共建项目（LHGJ20190751）

Effects of Mtb-infected type Ⅱ alveolar epithelial cells-derived exosome on the polarization of macrophages through miR-145

LI Jianjun, WU Sufang, BAI Fengxi   

  1. Department of Tuberculosis Ⅱ, Henan Chest Hospital, Zhengzhou 450008, China
  • Received:2022-01-26 Revised:2022-03-18 Published:2022-07-15 Online:2022-07-15

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摘要： 目的　探讨结核分枝杆菌（Mtb）感染Ⅱ型肺泡上皮细胞（AECⅡ）来源外泌体（Exos）对巨噬细胞极化的影响。方法　实验1：体外培养AECⅡ和Mtb毒株H37Rv，用Mtb感染AECⅡ并分离Exos，分为AECⅡ组和Mtb-AECⅡ组；透射电子显微镜（TEM）和纳米粒子追踪分析（NTA）观察Exos形态，检测Exos的数量和大小；流式细胞术鉴定Exos表面特征标志物CD63、CD81和HSP70表达；BCA法检测Exos的蛋白质含量；miRNA微阵列检测2组间差异miRNA表达谱并进行验证；酶联免疫吸附试验（ELISA）检测细胞培养上清液中肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6和IL-10水平。实验2：将AECⅡ分为对照组、mimic-NC组、miR-145 mimic组、inhibitor-NC组、miR-145 inhibitor组，用过表达或沉默miR-145的慢病毒转染AECⅡ后用Mtb感染并分离Exos，实时荧光定量PCR（qPCR）检测转染后Mtb-AECⅡ Exos中miR-145水平；流式细胞术检测巨噬细胞M1型标志物（CD86）、M2型标志物（CD163）表达；qPCR检测巨噬细胞中miR-145、TNF-α、诱导型一氧化氮合酶（iNOS）、精氨酸酶1（Arg-1）和IL-10的mRNA水平。结果　实验1：Mtb感染的AECⅡ分泌Exos为球形囊泡，直径约为100 nm；Exos中CD63、CD81、HSP70均呈阳性；与AECⅡ组相比，Mtb-AECⅡ组中Exos的数量和蛋白质含量、miR-145、IL-10水平增加（P＜0.05），TNF-α和IL-6水平降低（P＜0.05）。实验2：与对照组相比，miR-145 mimic组Mtb-AECⅡ Exos中miR-145水平、CD163阳性巨噬细胞百分比及巨噬细胞中miR-145、Arg-1和IL-10 mRNA水平升高（P＜0.05），CD86阳性巨噬细胞百分比、巨噬细胞中TNF-α mRNA、iNOS mRNA水平降低（P＜0.05），miR-145 inhibitor组上述指标的变化与miR-145 mimic组相反。结论　Mtb感染AECⅡ来源Exos可能通过miR-145刺激巨噬细胞向M2型极化并抑制M1型极化，对抗炎症。

关键词: 结核分枝杆菌, 巨噬细胞, 肺泡, 肺泡上皮细胞, 外泌体, 基因表达调控, Ⅱ型肺泡上皮细胞, microRNA-145

Abstract: Objective　To investigate the effect of Mycobacterium tuberculosis (Mtb)-infected type Ⅱ alveolar epithelial cells (AECⅡ)-derived exosomes (Exos) on the polarization of macrophages. Methods　Experiment 1: AECⅡ and Mtb strain H37Rv were cultured in vitro. AECⅡ was infected with Mtb and Exos, and was isolated. Exos were divided into the AECⅡ group and the Mtb-AECⅡ group. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to observe the morphology of Exos and to measure the number and size of Exos. Flow cytometry was used to identify the expressions of Exos surface characteristic markers CD63, CD81 and HSP70. BCA method was used to measure the protein content of Exos. miRNA microarray was used to detect and verify the differential miRNA expression profile between the two groups. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and IL-10 in the cell culture supernatant. Experiment 2: AECⅡ was separated into the control group, the mimic-NC group, the miR-145 mimic group, the inhibitor-NC group and the miR-145 inhibitor group. After AECⅡ was transfected with lentivirus that overexpressed or silenced miR-145, AECⅡ was infected by Mtb, and then Exos were isolated. Real-time fluorescence quantitative PCR (qPCR) was used to measure the level of miR-145 in Mtb-AECⅡ Exos after transfection. Flow cytometry was used to detect the expression levels of M1 type marker (CD86) and M2 type marker (CD163) of macrophages, and qPCR was used to detect the levels of miR-145, TNF-α, inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1) and IL-10 mRNAs in macrophages. Results　Experiment 1: Mtb-infected AEC Ⅱ secreted Exos as spherical vesicles with a diameter of about 100 nm. CD63, CD81 and HSP70 were all positive in Exos. Compared with the AECⅡ group, the quantity and protein content of Exos, levels of miR-145 and IL-10 were increased in the Mtb-AECⅡ group (P＜0.05), and levels of TNF-α and IL-6 were decreased (P＜0.05). Experiment 2: compared with the control group, the level of miR-145 in Mtb-AECⅡ Exos, the percentage of CD163-positive macrophages, the levels of miR-145, Arg-1 mRNA and IL-10 mRNA in macrophages were increased in the miR-145 mimic group (P＜0.05), and the percentage of CD86-positive macrophages, the levels of TNF-α mRNA and iNOS mRNA in macrophages were reduced (P＜0.05). Changes of the above indicators in the miR-145 inhibitor group were opposite to those in the miR-145 mimic group. Conclusion Mtb infection with AECⅡ-derived Exos may stimulate M2 type polarization of macrophages and inhibit M1 polarization through miR-145, thus resisting inflammation.

Key words: mycobacterium tuberculosis, macrophages, alveolar, alveolar epithelial cells, exosomes, gene expression regulation, type Ⅱ alveolar epithelial cells, microRNA-145