树突状细胞来源外泌体通过NF-κB通路影响内皮细胞致促炎因子的变化

Effect of dendritic cell-derived exosomes on endothelial pro-inflammatory factors through NF-κB pathway

目的探讨来源于树突状细胞(DCs)的外泌体对人脐静脉内皮细胞(HUVEC)炎症因子的作用和机制。方法从C57BL/6J小鼠中分离获得骨髓树突状细胞(BMDCs)并培养,采用超速离心法分别从BMDCs及经脂多糖(LPS)诱导成熟的BMDCs培养基中分离获得外泌体、并经电镜扫描及免疫印迹法(WB)验证;构建CD63-GFP慢病毒载体并转染BMDCs细胞,Transwell共培养BMDCs细胞与HUVEC、并用外泌体处理细胞;应用ELISA法检测各组细胞肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)的浓度,蛋白质印迹法(Western blot)测试p100和p105的磷酸化水平,实时荧光定量聚合酶链式反应(RT-PCR)测试TNF-α和IL-6的mRNA表达水平,利用免疫荧光技术检测HUVEC摄取外泌体的情况。结果激光共聚焦显微镜观察显示,HUVEC能摄取BMDCs来源的外泌体;通过LPS诱导的成熟和未成熟的BMDCs外泌体中的IL和TNF水平无统计学差异;BMDCs与HUVEC共同培养的培养基中IL-6和TNF-α的浓度和p100和p105的磷酸化水平比单纯培养HUVEC培养基中的表达明显升高(P<0.05),但BMDCs外泌体被外泌体抑制剂GW4869抑制后,TNF-α和IL-6含量及p100和p105的磷酸化水平明显下降(P<0.01);共培养联合NF-κB抑制剂BAY11-7082组中的TNF-α和IL-6含量及p100和p105磷酸化水平比单纯HUVEC与BMDCs共培养组明显下降(P<0.001)。结论BMDCs来源的外泌体能够被HUVEC摄取,并促进HUVEC炎症因子TNF-α和IL-6升高,其机制可能与BMDCs来源的外泌体影响NF-κB信号通路中p100以及p105的磷酸化水平有关。

Objective To investigate the effect and mechanism of exosomes derived from dendritic cells(DCs)on the pro-inflammatory factors from endothelial cells by NF-κB pathway.Methods Bone marrow dendritic cells(BMDCs)were isolated from C57BL/6J mice,exosome from BMDCs and LPS-induced mature BMDCs culture medium were isolated by ultracentrifugation.Exosomes were then verified with electron microscopy scanning and Western blot.CD63-GFP fusion lentiviral vector was constructed and transfected into BMDCs.BMDCs and human umbilical vein endothelial cells(HUVEC)were co-cultured by Transwell,and the isolated exosomes were treated with the cells.Then,the protein level of tumor necrosis factor(TNF-α)and interleukin(IL-6)were quantified by ELISA,phosphorylation of p100 and p105 were detected by Western blot,mRNA level of TNF-αand IL-6 were explored by qRT-PCR;exosomes absorption by HUVEC was observed by immunofluorescence.Results Confocal microscopy showed that exosomes from BMDCs could be absorbed by HUVEC.There was no significant difference in TNF-αand IL-6 between mature BMDCs induced by LPS and immature BMDCs exosomes(P>0.05).Compared with HUVEC alone,the contents of TNF-α,IL-6 and the phosphorylation levels of p100 and p105 in HUVEC and BMDCs co-cultured medium were significantly increased(P<0.05),while the contents of TNF-αand IL-6 and the phosphorylation levels of p100 and p105 were significantly decreased after GW4869 inhibited the secretion of exosomes of BMDCs(P<0.01);compared with the co-culture group of HUVEC and BMDCs,the co-culture group combined with NF-κB showed the contents of TNF-α,IL-6,and the phosphorylation levels of p100 and p105 were decreased in BAY11-7082 group(P<0.001).Conclusions BMDCS-derived exosomes can be absorbed by HUVEC and activate phosphorylation of p100 and p105 through NF-κB signaling pathway,thereby increasing the expression and secretion of TNF-αand IL-6.