BAFF调节免疫性血小板减少症模型小鼠的Th17/Treg平衡的研究
Regulation of Th17/Treg Balance by BAFF in a Mouse Model of Immune Thrombocytopenia
李巍 1马西虎 1刘晓 1费飞 1秦兰 1买尔吾甫·木合布力 2白玉盛1
作者信息
- 1. 新疆医科大学第四附属中医医院血液科,新疆 乌鲁木齐 830001
- 2. 新疆医科大学,新疆 乌鲁木齐 830054
- 折叠
摘要
目的:探讨B细胞激活因子(BAFF)对免疫性血小板减少症模型小鼠体内辅助性T细胞17(Th17)/调节性T 细胞(Treg)平衡的调节作用和潜在机制.方法:制备豚鼠抗小鼠血小板抗血清(GP-APS),并将150 只无特定病原级别的成年雄性 BALB/c 小鼠(7～8 周龄)随机分为 5 组,每组 30只.分别为对照组(空白对照)和ITP 组(GP-APS 诱导),ITP+rhBAFF 组(ITP 组联合静脉注射 50μg/kg/50μL重组人BAFF蛋白),并在ITP+rhBAFF组处理的基础上分别联合 Notch1 的抑制剂(DAPT)或PI3K/Akt的抑制剂Polygalacin D(PGD),设立ITP+rhBAFF+DAPT组和ITP+rhBAFF+PGD组,除对照组和ITP 组外,均为静脉注射给药,DAPT注射剂量100μg/kg;PGD注射剂量25μg/kg,静脉注射总体积均为50μL,每日1 次.1 周后取小鼠1mL外周血并分离血清和单个核细胞.用免疫荧光化学检测单个核细胞中BAFF和Notch1 的定位.对外周血中的血小板进行计数.酶联免疫吸附法(ELSIA)检测小鼠外周血血清 BAFF 的水平.Western blot 检测小鼠外周血单个核细胞中 PI3K、AKT、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的蛋白表达.流式细胞术检测单个核细胞中Th17/Treg 的比例变化.结果:ITP 小鼠外周血单个核细胞的BAFF与Notch1 共定位在细胞膜.与对照组比较,ITP 组BAFF、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的表达增加,血小板数目和 Treg 比例减少,Th17 比例增加(P＜0.05).与ITP 组比较,ITP+rhBAFF组BAFF、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的表达增加,血小板数目和Treg比例减少,Th17 比例增加(P＜0.05).与 ITP+rhBAFF 组比较,ITP+rhBAFF+DAPT 组 BAFF、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的表达降低,血小板数目和 Treg 比例增加,Th17 比例降低(P＜0.05).与ITP+rhBAFF组比较,ITP+rhBAFF+PGD组BAFF、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的表达降低,血小板数目和 Treg 比例增加,Th17 比例降低(P＜0.05).结论:BAFF 通过激活 Notch1/PI3K/Akt信号通路促进免疫性血小板减少症模型小鼠体内Th17 比例增加及Treg比例减少.
Abstract
Objective:To explore the regulatory role of B-cell activating factor(BAFF)on the balance of helper T cell 17(Th17)and regulatory T cell(Treg)in vivo in a mouse model of immune thrombocytope-nia(ITP).Methods:Guinea pig anti-mouse platelet serum(GP-APS)was prepared,and 150 adult male BALB/c mice(7-8 weeks old)were randomly divided into 5 groups(n=30 per group):control(blank con-trol),ITP(induced by GP-APS),ITP+rhBAFF(ITP mice treated with intravenous injection of recombinant human BAFF protein at 50μg/kg/50μL),ITP+rhBAFF+DAPT(combined with Notch1 inhibitor DAPT),and ITP+rhBAFF+PGD(combined with PI3K/Akt inhibitor Polygalacin D).DAPT was injected at 100μg/kg,and PGD at 25 μg/kg,both with a total volume of 50μL and administered once daily.Peripheral blood(1mL)was collected from mice after 1 week for serum and mononuclear cell isolation.Immunofluorescence staining was used to detect BAFF and Notch1 localization in mononuclear cells.Platelet counts in peripheral blood were determined.Enzyme-linked immunosorbent assay(ELISA)was performed to measure BAFF lev-els in mouse serum.Western blotting was conducted to assess protein expression of PI3K,AKT,Notch1,p-Akt(Thr308),and p-Akt(Ser473)in mononuclear cells.Flow cytometry was used to analyze changes in Th17/Treg ratio in mononuclear cells.Results:BAFF and Notch1 were co-localized on the cell membrane of mononuclear cells in peripheral blood of ITP mice.Compared with the control group,ITP group showed in-creased expression of BAFF,Notch1,p-Akt(Thr308),p-Akt(Ser473),increased Th17 proportion,de-creased platelet count,and decreased Treg proportion(P＜0.05).Compared with the ITP group,ITP+rh-BAFF group exhibited increased BAFF,Notch1,p-Akt(Thr308),p-Akt(Ser473)expression,increased Th17 proportion,decreased platelet count,and decreased Treg proportion(P＜0.05).Compared with the ITP+rhBAFF group,both ITP+rhBAFF+DAPT and ITP+rhBAFF+PGD groups showed decreased BAFF,Notch1,p-Akt(Thr308),p-Akt(Ser473)expression,increased platelet count,increased Treg proportion,and de-creased Th17 proportion(P＜0.05).Conclusion:BAFF promotes an increase in Th17 proportion and de-crease in Treg proportion in a mouse model of immune thrombocytopenia by activating the Notch1/PI3K/Akt signaling pathway.
关键词
B细胞激活因子/免疫性血小板减少症/小鼠/辅助性T细胞17/调节性T细胞的平衡Key words
B-cell activating factor/Immune thrombocytopenia/Mouse/Th17/regulatory T cell balance引用本文复制引用
基金项目
新疆维吾尔自治区自然科学基金项目(2022D01C167)
出版年
2024
国家科技期刊平台
NSTL
万方数据