何洋,安天志,李成,周石,谢坪,段庆红
HE Yang,AN Tianzhi,LI Cheng,ZHOU Shi,XIE Ping,DUAN Qinghong

• 1:贵州医科大学影像学院
• 2:四川省人民医院介入科

摘要(Abstract)：

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 LPSinduced 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-α and 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-α and 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-6and 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-α and 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-α and IL-6.

关键词(KeyWords)： 骨髓树突状细胞;外泌体;NF-κB通路;人脐静脉内皮细胞;细胞因子
bone marrow dendritic cells (BMDCs);exosome;NF-κB pathway;human umbilical vein endothelial cell (HUVEC);cytokine

Abstract:

Keywords:

基金项目(Foundation): 贵州省科技计划项目（黔科合基础[2019]1259）;; 四川省科技厅重点研发项目（2020YFS0405）

作者(Author): 何洋,安天志,李成,周石,谢坪,段庆红
HE Yang,AN Tianzhi,LI Cheng,ZHOU Shi,XIE Ping,DUAN Qinghong

DOI: 10.19367/j.cnki.2096-8388.2022.06.006

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