HBV诱导生成的外泌体对巨噬细胞表型和功能的影响  

Effects of HBV-induced exosomes on macrophage phenotype and function

作　　者：慕容容 李爽 叶海艳[1] 陈利民[1] 李玉佳[1] 李世林[1] MU Rongrong;LI Shuang;YE Haiyan;CHEN Limin;LI Yujia;LI Shilin(Institute of Blood Transfusion,Chinese Academy of Medical Science and Peking Union Medical College,Chengdu 610052,China;Shanghai University of Medicine&Health Sciences)

机构地区：[1]中国医学科学院北京协和医学院输血研究所,四川成都610052 [2]上海健康医学院

出　　处：《中国输血杂志》2022年第5期488-493,共6页Chinese Journal of Blood Transfusion

基　　金：四川省科技厅国际合作项目(2021YFH0100)。

摘　　要：目的 探究感染乙肝病毒(HBV)的肝细胞产生的外泌体对巨噬细胞细胞表型和功能的影响。方法 超速离心法结合免疫吸附法收集能够产生HBV颗粒的HepAD38细胞和普通肝癌细胞HepG2分泌的外泌体,通过粒径分析(NTA)、扫描电子显微镜及Western blot检测所提取外泌体的质量和纯度。人单核细胞(THP-1)经PMA诱导后分化为M0巨噬细胞并加入外泌体刺激,在不同时间点分别收取RNA和蛋白样本,实时荧光定量PCR(qRT-PCR)检测细胞因子mRNA表达水平,酶联免疫吸附测定法(ELISA)检测细胞因子蛋白表达水平;同时,用中性红实验检测巨噬细胞胞饮功能,用试剂盒检测巨噬细胞活性氧(ROS)产生水平。对提取纯化后的外泌体进行人逆转录芯片检测,选取差异表达的microRNA,通过qRT-PCR进行进一步验证。结果 与HepG2来源的外泌体相比,HepAD38细胞产生的外泌体可以引起巨噬细胞IL-1β、MCP-1及TNFα表达水平明显升高但对巨噬细胞胞饮功能和ROS生成量无明显影响。人逆转录芯片检测结果经KEGG分析及qRT-PCR验证,发现miR-6824-3p在高表达后也可引起IL-1β、MCP-1及TNFα表达水平明显升高。结论 感染HBV的HepAD38细胞可以通过外泌体影响巨噬细胞炎症因子的表达,而外泌体中的miR-6824-3p可能在这个过程中发挥重要作用。Objective To investigate the effect of exosomes produced by hepatitis B virus(HBV)-infected cells on the phenotype and function of macrophages.Methods The exosomes secreted by HepAD38 cells, which were capable of producing HBV and HepG2 cells, were collected by ultracentrifugation combined with immunosorbent method.The quality and purity of the extracted exosomes were verified by nanoparticle tracking analysis(NTA),scanning electron microscope and Western blot.The M0 THP-1 macrophages differentiated by PMA were stimulated by HepAD38 derived-or HepG2 derived exosomes.Total RNA and protein samples were collected at different time points after stimulation.Real-time fluorescence quantitative PCR(qRT-PCR) and enzyme-linked immunosorbent assay(ELISA) were used to detect cytokine mRNA and protein expressions, respectively.Meanwhile, neutral red assay was performed to analyze macrophage pinocytosis activity, and a commercial kit was used to measure reactive oxygen species(ROS) in THP-1 macrophages.Human reverse transcription chip detection was performed to obtain the microRNAs profile of the exosomes.And the effect of selected miRNA on macrophages was further confirmed by qRT-PCR.Results Compared with HepG2-derived exosomes, HepAD38-derived exosomes increased the mRNA and protein expressions of IL-1β,MCP-1 and TNFα significantly.However, no difference of pinocytosis capacity or ROS production was found between the HepAD38-derived exosomes group and HepG2-derived exosomes group.Human reverse transcription chip detection results were verified by KEGG analysis and qRT-PCR,and it was found that miR-6824-3 p could also significantly increase the expression levels of IL-1β,MCP-1 and TNFα after high expression.Conclusion This study found that exosomes produced by HepAD38 cells may stimulate macrophages to produce inflammatory factors such as IL-1β,MCP-1 and TNFα through miR-6824-3 p, thereby playing a role in HBV infection.

关 键 词：外泌体 HBV 巨噬细胞 细胞因子 巨噬细胞功能 

分 类 号：R457.1[医药卫生—治疗学]