EBV miR-BART17-3p对原发免疫性血小板减少症患儿Treg/Th17平衡的影响  

Effect of EBV miR-BART17-3p on Treg/Th17 Balance in Children with Primary Immune Thrombocytopenia

作　　者：张雅丽 闫雪峰 冯茹清 ZHANG Yali;YAN Xuefeng;FENG Ruqing(Zhangjiakou Maternal and Child Health Hospital,Zhangjiakou,Hebei,China 075000)

机构地区：[1]河北省张家口市妇幼保健院,河北张家口075000

出　　处：《中国药业》2024年第18期51-57,共7页China Pharmaceuticals

基　　金：河北省张家口市重点研发计划项目[1921068D]。

摘　　要：目的探讨EBV mi R-BART17-3p影响原发免疫性血小板减少症(ITP)患儿Treg/Th17平衡的机制。方法收集ITP患儿(ITP组,20例)和健康儿童(对照组,20例)外周血并分离CD_(4)^(+)T细胞。采用实时荧光定量聚合酶链式反应法、Western blot法、酶联免疫吸附法检测EBV mi R-BART17-3p、T细胞免疫球蛋白黏蛋白3(Tim-3)、叉头框蛋白P3(Fox P3)、白细胞介素17A(IL-17A)和转化生长因子-β(TGF-β)的m RNA、蛋白表达水平及含量。采用双荧光素酶报告基因实验考察EBV mi R-BART17-3p对Tim-3表达水平的影响。将15只BALB/C小鼠随机分为空白对照组、模型组、观察组,各5只。腹腔注射抗血小板抗体MWReg30以复制ITP小鼠模型,建模4 d后观察组小鼠予尾静脉注射携带EBV mi R-BART17-3p inhibitor的腺病毒载体。细胞染色并观察形态,检测外周血中TGF-β、IL-17A含量及血小板计数,采用流式细胞仪分别检测CD_(4)^(+)T细胞中Th17和Treg水平,并计算二者百分比。结果与对照组比较,ITP组患儿外周血EBV mi R-BART17-3p表达水平显著升高,Tim-3和TGF-βm RNA表达水平显著降低(P<0.05);Tim-3 m RNA表达水平与EBV mi R-BART17-3p表达水平呈显著负相关(r=-0.732,P<0.001)。Tim-3慢病毒载体p LKO.1-sh-Tim-3(sh-Tim-3)可显著降低Tim-3、Fox P3、TGF-β水平(P<0.05)。mi R-BART17-3p mimic显著升高了CD_(4)^(+)T细胞中mi R-BART17-3p的表达水平,并显著降低了Tim-3、Fox P3、TGF-βm RNA和蛋白表达水平(P<0.05);mi R-BART17-3p mimic可显著降低TGF-β含量,Tim-3+mi R-BART17-3p过表达逆转了mi R-BART17-3p mimic对TGF-β的抑制作用。动物实验结果显示,沉默EBV mi R-BART17-3p可促进Treg分化,减少脾脏和骨髓组织中的巨核细胞计数,并显著增加外周血中血小板计数。结论EBV mi R-BART17-3p可通过Fox P3/Tim-3途径调节ITP患儿Treg/Th17的免疫失衡。Objective To investigate the mechanism of EBV miR-BART17-3p affecting the Treg/Th17 balance in children with primary immune thrombocytopenia(ITP).Methods The peripheral blood from ITP children(ITP group,20 cases)and healthy children(control group,20 cases)was collected and the CD_(4)^(+)T cells were isolated.The real-time fluorescence quantitative polymerase chain reaction,Western blot,and enzyme-linked immunosorbent assay were used to detect the mRNA,protein expression levels and contents of EBV miR-BART17-3p,T cell immunoglobulin domain and mucin domain-3(Tim-3),forkhead box protein P3(FoxP3),interleukin-17A(IL-17A),and transforming growth factor-β(TGF-β).The dual-luciferase reporter gene assay was used to explore the effect of EBV miR-BART17-3p on the expression level of Tim-3.Fifteen BALB/C mice were randomly divided into the blank control group,the model group and the observation group,with five mice in each group.Mice were intraperitoneally injected with antiplatelet antibody MWReg30 to construct the ITP models.After 4 d of modeling,the mice in the observation group were injected with adenovirus vector carrying EBV miR-BART17-3p inhibitor via the tail vein.Cells were stained for morphological observation;the TGF-β,IL-17A contents and platelet count in peripheral blood were detected;the flow cytometry was used to measure the Th17 and Treg levels in CD_(4)^(+)T cells,and their percentage was calculated.Results Compared with those in the control group,the expression level of EBV miR-BART17-3p in peripheral blood in the ITP group significantly increased,while the expression levels of Tim-3 and TGF-βmRNA significantly decreased(P<0.05);the expression level of Tim-3 mRNA was significantly negatively correlated with the expression level of EBV miR-BART17-3p(r=-0.732,P<0.001).Tim-3 lentivirus vector carrying pLKO.1-sh-Tim-3(sh-Tim-3)could significantly decrease the Tim-3,FoxP3 and TGF-βlevels(P<0.05).The miR-BART17-3p mimic significantly increased the expression levels of miR-BART17-3p in CD_(4)^(+)T cells,and

关 键 词：EBV mi R-BART17-3p T细胞免疫球蛋白黏蛋白3 叉头框蛋白P3 辅助性T细胞17 原发性血小板减少症 

分 类 号：R917[医药卫生—药物分析学] R927[医药卫生—药学]