新生儿败血症患儿外周血CD4^(+)T细胞miR-146a-3p降低、IL-17水平增加  

miR-146a-3p is down-regulated and IL-17 is up-regulated in peripheral blood CD4^(+) T cells of children with neonatal sepsis

作　　者：冯燕妮[1] 罗宏成[2] 姚小敏 杨松媚[1] 黄艳[1] 陈娟娟 刘运强[1] 农美杏 梁炎贤 梁玉美[1] FENG Yanni;LUO Hongcheng;YAO Xiaomin;YANG Songmei;HUANG Yan;CHEN Juanjuan;LIU Yunqiang;NONG Meixing;LIANG Yanxian;LIANG Yumei(Department of Neonatology,Affiliated Hospital of Youjiang Medical University for Nationalities,Baise 533000,China;Department of Clinical Laboratory,Affiliated Hospital of Youjiang Medical University for Nationalities,Baise 533000,China)

机构地区：[1]右江民族医学院附属医院新生儿科,广西百色533000 [2]右江民族医学院附属医院检验科,广西百色533000

出　　处：《细胞与分子免疫学杂志》2021年第12期1120-1127,共8页Chinese Journal of Cellular and Molecular Immunology

基　　金：广西壮族自治区第五批自治区本级财政科技计划项目(2019JJB140023);广西壮族自治区卫生厅自筹经费科研课题(Z2011145)。

摘　　要：目的研究新生儿败血症患儿外周血CD4^(+)T细胞微小RNA-146a-3p(miR-146a-3p)、白细胞介素17(IL-17)的表达及机制。方法应用实时定量PCR检测66例败血症患儿、40例感染性疾病患儿及40例健康新生儿(对照组)CD4^(+)T细胞miR-146a-3p、IL-17 mRNA的表达,ELISA检测外周血IL-17水平。双荧光素酶报告实验验证miR-146a-3p对IL-17的靶向作用关系。分离CD4^(+)T细胞,采用miR-146a-3p模拟物(miR-146a-3p mimic)或miR-146a-3p抑制物(miR-146a-3p inhibitor)促进或抑制CD4^(+)T细胞miR-146a-3p表达后,流式细胞术检测Th17细胞比例变化,甲基化特异PCR检测miR-146a-3p基因甲基化情况。CD4^(+)T细胞中加入甲基化抑制剂5-氮杂-2′-脱氧胞嘧啶(5-Aza-dC)后,观察Th17细胞比例变化及IL-17表达变化。结果与感染性疾病患儿及对照组相比,败血症组患儿外周血CD4^(+)T细胞miR-146a-3p表达降低,IL-17 mRNA水平增加,外周血IL-17水平升高。CD4^(+)T细胞中转染miR-146a-3p mimic能够显著抑制IL-173′非翻译区(3′UTR)野生型的荧光素酶活性。转染miR-146a-3p mimic后,CD4^(+)T细胞IL-17 mRNA显著降低,培养上清中IL-17水平和Th17细胞比例显著降低,而转染miR-146a-3p inhibitor后,IL-17 mRNA表达显著升高,上清中IL-17水平显著升高,Th17细胞比例升高。败血症组外周血miR-146a-3p基因启动子甲基化阳性率明显高于对照组及感染组,CD4^(+)T细胞中加入5-Aza-dC后,miR-146a-3p表达升高,IL-17 mRNA表达降低,上清中IL-17水平降低,Th17细胞比例明显降低。结论新生儿败血症患儿外周血CD4^(+)T细胞miR-146a-3p表达下调,IL-17表达上调。Objective To investigate the expression of microRNA-146a-3p(miR-146a-3p)and interleukin 17(IL-17)in peripheral blood CD4^(+)T cells of neonates with sepsis(NS)and its regulation mechanism.Methods The expression of miR-146a-3p and IL-17 mRNA in CD4^(+)T cells of 66 children with sepsis(septicemia group),40 children with infectious diseases(infection group),and 40 healthy newborns(control group)were detected by real-time quantitative PCR,and the level of IL-17 in peripheral blood was detected by ELISA.The targeting effect of the miR-146a-3p on IL-17 was verified by the dual luciferase reporter assay.After isolation of the CD4^(+)T cells,the expression of miR-146a-3p in CD4^(+)T cells was promoted or inhibited by miR-146a-3p mimic or miR-146a-3p inhibitor.The proportion of Th17 cells was detected by flow cytometry,and the methylation of miR-146a-3p gene was detected by methylation specific PCR.The changes of Th17 cell proportion and IL-17 expression were observed after adding methylation inhibitor 5-Aza-2′-deoxycytidine(5-Aza-dC)to CD4^(+)T cells.Results Compared with those in the infection group and the control group,the expression of miR-146a-3p in peripheral blood CD4^(+)T cells decreased,while the expression of IL-17 mRNA and the level of IL-17 in peripheral bloodincreased in septicemia group.Transfection of miR-146a-3p mimic in CD4^(+)T cells significantly inhibited the activity of wild-type luciferase in the 3′UTR of IL-17.After transfection of miR-146a-3p mimic,the expression of IL-17 mRNA in CD4^(+)T cells,and the level of IL-17 and the proportion of Th17 cells in the supernatant were significantly decreased.After transfection of miR-146a-3p inhibitor,the expression of IL-17 mRNA,and the level of IL-17 and the proportion of Th17 cells in the supernatant were increased.The methylation rate of miR-146a-3p gene promoter in the peripheral blood of the septicemia group was significantly higher than those of the control group and the infection group.After the addition of 5-Aza-dC in CD4^(+)T cells,the expressi

关 键 词：新生儿败血症 微小RNA-146a-3p(miR-146a-3p) 白细胞介素17(IL-17) 

分 类 号：R722.1[医药卫生—儿科] R392.12[医药卫生—临床医学]