PRDM1调控NF-κB信号通路对巨噬细胞极化和功能的影响  

Effect of PRDM1 on macrophage polarization and function via regulating NF-κB signaling pathway

作　　者：张梦莹 李志 李雪琴 钟民 吕坤 ZHANG Meng-Ying;LI Zhi;LI Xue-Qin;ZHONG Min;LYU Kun(Central Laboratory of the first Affiliated Hospital of Wannan Medical College,Yijishan Hospital,Wuhu 241001,China)

机构地区：[1]皖南医学院第一附属医院弋矶山医院中心实验室,芜湖241001 [2]皖南医学院第一附属医院弋矶山医院风湿免疫科,芜湖241001

出　　处：《中国免疫学杂志》2021年第11期1286-1291,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金(81772180,81701557);皖南医学院重点科研项目培育基金(WK2017ZF02)资助。

摘　　要：目的:本文旨在探讨正性调节区锌指蛋白1(PRDM1)对小鼠骨髓来源巨噬细胞(BMDMs)极化和功能的影响及其分子机制。方法:体外提取BMDMs作为研究对象,并进行极化诱导,采用荧光定量PCR和Western blot法检测不同极化状态的巨噬细胞PRDM1的表达水平;设计构建PRDM1过表达腺病毒载体,瞬时感染BMDMs,48 h后分别加入IFN-γ/LPS或IL-4刺激诱导为M1/M2型巨噬细胞,采用荧光定量PCR分别检测M1/M2的标志基因iNOS、IL-12、TNF-α、Arg1、YM-1、FIZZ1的表达;ELISA法检测各组细胞上清液炎症因子TNF-α、IL-12和IL-13的表达量;细菌杀伤试验和吞噬试验检测PRDM1过表达对巨噬细胞功能的影响;构建巨噬细胞M2极化模型,将PRDM1过表达腺病毒感染细胞,Western blot法检测核转录因子-κB(NF-κB)信号通路中的下游信号分子p65及IKK蛋白的总体水平及磷酸化水平。结果:荧光定量PCR和Western blot结果显示,M1型巨噬细胞的PRDM1表达水平显著高于M2型巨噬细胞;与阴性对照组相比,过表达PRDM1明显促进巨噬细胞M1型标志分子iNOS、IL-12及TNF-α的表达,能抑制M2型标志分子Arg1、YM-1及FIZZ1的表达;并且显著增强BMDMs的杀菌活性,而降低了BMDMs的吞噬功能。在M2型巨噬细胞中过表达PRDM1后,NF-κB信号通路中p65及IKK磷酸化水平较阴性对照组显著升高。结论:NF-κB信号通路可能参与PRDM1调控巨噬细胞的M1极化。Objective:The purpose of this study was to investigate the effect and molecular mechanism of PRDM1 on the polarization and function of bone marrow-derived macrophages(BMDMs).Methods:BMDMs were extracted in vitro as research objects and polarization induction was performed.RT-qPCR and Western blot were used to detect the expression level of PRDM1 in M1/M2 macrophages. We next constructed PRDM1 gene overexpression adenovirus vector and infected BMDMs,after 48 h culture,IFN-γ/LPS or IL-4 were added to induce M1/M2 macrophages. Then,we used RT-qPCR to detect the mRNA expression levels of iNOS,IL-12,TNF-α or Arg1,YM-1,FIZZ1 which was the marker genes of M1 and M2,respectively. ELISA was used to detect the inflammatory factor secretion of TNF-α,IL-12,IL-13 in the cell supernatant of PRDM1 overexpression group and control. Bactericidal activity assay and phagocytosis assay were used to detect the effect of PRDM1 overexpression on macrophage function. Moreover,we detected the total protein and phosphorylation protein levels of p65 and IKK which was the key molecules of nuclear factor-κB(NF-κB)signaling pathway in M2 macrophages with PRDM1 overexpression by Western blot.Results:The results of RT-qPCR showed that PRDM1 gene was highly expressed in M1 macrophages compared to M2. Upon overexpression of the PRDM1 expression and compared to the NC group,the M1 macrophages induced by LPS and IFN-γ showed increased mRNA expression levels of iNOS,TNF-α,and IL-12,while M2 macrophages polarized by IL-4 showed decreased mRNA expression levels of Arg1,YM1,and FIZZ1. Furthermore,compared to control,PRDM1 overexpression group could produce higher levels of pro-inflammatory cytokines TNF-α and IL-12,which the secretion level of anti-inflammatory cytokine IL-13 was decreased. Bactericidal activity assay showed that overexpression of PRDM1 significantly strengthen the bactericidal activity of BMDMs,while reducing the phagocytic funtion of BMDMs. Overexpression of PRDM1 in M2 macrophages,the phosphorylation levels of p65 and IKK we

关 键 词：巨噬细胞极化 PRDM1基因 核转录因子ΚB 

分 类 号：R392.11[医药卫生—免疫学]