南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (5): 658-664.doi: 10.12122/j.issn.1673-4254.2022.05.05

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干扰肿瘤相关巨噬细胞的P2X4受体表达可抑制胶质瘤细胞的迁移和侵袭

杨学智，沈 红，李 群，代自超，杨荣强，黄国宾，陈 蕊，王 芳，宋精玲，华海蓉   

  1. 病理学与病理生理学系，临床技能中心，口腔医学研究所，附属口腔医院，电子显微镜室，昆明医科大学，云南 昆明 650500；昭通市第一人民医院病理科，云南 昭通 657099
  • 出版日期:2022-05-20 发布日期:2022-06-02

Interference of P2X4 receptor expression in tumor-associated macrophages suppresses migration and invasion of glioma cells

YANG Xuezhi, SHEN Hong, LI Qun, DAI Zichao, YANG Rongqiang, HUANG Guobin, CHEN Rui, WANG Fang, SONG Jingling, HUA Hairong   

  1. Department of Pathology and Pathophysiology, Clinic Skill Center, Institute of Stomatology, Affiliated Stomatology Hospital, Electron Microscope, Kunming Medical University, Kunming 650500, China; Department of Pathology, Zhaotong First People's Hospital, Zhaotong 657099, China
  • Online:2022-05-20 Published:2022-06-02

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摘要： 目的 研究P2X4受体在小鼠胶质瘤中肿瘤相关巨噬细胞（TAMs）的表达对胶质瘤细胞侵袭迁移的影响及分子机制。方法 将16只健康C57BL/6小鼠随机分为肿瘤组、对照组（8只/组），利用小鼠胶质瘤GL261细胞接种于小鼠大脑尾状核，建立小鼠脑胶质瘤模型。术后第21天处死小鼠，取荷瘤小鼠及正常小鼠脑组织，采用HE染色观察小鼠胶质瘤形态，利用免疫荧光检测Iba-1、 P2X4受体的表达情况；应用GL261条件培养基将RAW264.7细胞诱导为TAMs，采用RT-qPCR检测巨噬细胞极化相关标志物及P2X4受体在TAMs的mRNA表达情况；采用Western blot检测P2X4受体在TAMs的蛋白表达情况；采用siRNA P2X4，下调TAMs中P2X4受体的蛋白表达，RT-qPCR、Western blot检测siRNA P2X4转染后TAMs中IL-1β、IL-18的mRNA及蛋白表达；利 用transwell侵袭及迁移实验检测siRNA P2X4 转染后TAMs对GL261细胞侵袭迁移能力的影响。结果 与对照组相比，荷瘤小鼠脑胶质瘤组织中有较高数量的Iba-1阳性细胞（P<0.0001），且P2X4受体在Iba-1阳性细胞中的表达增高（P=0.001）；使用GL261条件培养基刺激RAW264.7细胞转化为TAMs后，M2型巨噬细胞标志基因Arg-1、IL-10表达上调（P=0.0001、0.001），M1型巨噬细胞标志基因iNOS、TNF-α表达也上调（P=0.006、0.001），但以M2型巨噬细胞标志基因Arg-1、IL-10表达上调更为显著，同时TAMs中P2X4受体蛋白表达水平及mRNA水平均增高（P=0.005、0.014）。干扰TAMs中P2X4受体表达引起其IL-1β、IL-18的mRNA（P<0.01）及蛋白表达水平（P<0.01，P<0.05）降低，并抑制TAMs促进胶质瘤细胞侵袭和迁移的能力（P=0.004、0.017）。结论 降低肿瘤相关巨噬细胞P2X4受体表达可能通过IL-1β、IL-18影响胶质瘤细胞的迁移和侵袭。

关键词: 胶质瘤；P2X4受体；肿瘤相关巨噬细胞；侵袭；迁移

Abstract: Objective To investigate the effect of interference of P2X4 receptor expression in tumor-associated macrophages (TAMs) on invasion and migration of glioma cells. Methods C57BL/6 mouse models bearing gliomas in the caudate nucleus were examined for glioma pathology with HE staining and expressions of Iba-1 and P2X4 receptor with immunofluorescence assay. RAW264.7 cells were induced into TAMs using conditioned medium from GL261 cells, and the changes in mRNA expressions of macrophage polarization-related markers and the mRNA and protein expressions of P2X4 receptor were detected with RT-qPCR and Western blotting. The effect of siRNA-mediated P2X4 interference on IL-1β and IL-18 mRNA and protein expressions in the TAMs was detected with RT-qPCR and Western blotting. GL261 cells were cultured in the conditioned medium from the transfected TAMs, and the invasion and migration abilities of the cells were assessed with Transwell invasion and migration experiment. Results The glioma tissues from the tumor-bearing mice showed a significantly greater number of Iba-1-positive cells, where an obviously increased P2X4 receptor expression was detected (P=0.001), than the brain tissues of the control mice (P<0.001). The M2 macrophage markers (Arg-1 and IL-10) and M1 macrophage markers (iNOS and TNF-α) were both significantly up-regulated in the TAMs derived from RAW264.7 cells (all P<0.01), but the up-regulation of the M2 macrophage markers was more prominent; the expression levels of P2X4 receptor protein and mRNA were both increased in the TAMs (P<0.05). Interference of P2X4 receptor expression significantly lowered the mRNA（P<0.01）and protein （P<0.01, P<0.05）expression levels of IL-1β and IL-18 in the TAMs and obviously inhibited the ability of the TAMs to promote invasion and migration of the glioma cells (P<0.05). Conclusion Interference of P2X4 receptor in the TAMs suppresses the migration and invasion of glioma cells possibly by lowering the expressions of IL-1β and IL-18.

Key words: glioma; P2X4 receptor; tumor associated macrophages; invasion; migration