可诱导共刺激分子与程序性死亡-1在重症肌无力患者外周血表达的研究
Expression of costimulatory molecule inducible costimulator and coinhibitory molecule programmed death-1 in patients with myasthenia gravis
目的 检测重症肌无力(myasthenia gravis,MG)早期患者外周血中正性共刺激分子可诱导共刺激分子(inducible costimulator,ICOS)与负性共刺激分子程序性死亡-1(programmed death 1,PD-1)的表达,并探讨其临床意义.方法 分别收集2014年2月到2016年12月间在苏州大学附属第一医院就诊且确诊为MG患者82例外周血标本,作为试验组,同时收集20例非MG患者的其他免疫病患者(non-myasthenia gravis,NMG)及健康体检者56名的外周血标本作为对照组.应用免疫荧光标记、流式细胞术检测3组成员外周血单个核细胞膜上PD-1、ICOS及相应配体程序性死亡-1配体(PD-L1)、可诱导共刺激分子配体(ICOSL)的表达;同时收集2组患者及健康对照组血清,应用酶联免疫吸附法检测3组成员血清中可溶性PD-1(sPD-1)、可溶性PD-L1(sPD-L1)和细胞因子IL-4等的含量并进行统计学分析.结果 (1)流式分析结果:MG组CD4+T细胞上ICOS+亚群[57.29%(57.32%)]较健康对照组[15.6%(14.23%),Z=-6.560,P<0.05]及NMG组[23.4%(8.48%),Z=-3.645,P<0.05]显著增高,CD14+单核细胞上和CD19+B细胞上ICOSL+亚群较健康对照组和NMG组表达增高.MG组CD4+T细胞上PD-1+亚群[16.82%(10.66%)]较健康对照组[9.34%(9.18%),Z=-4.345,P<0.05]及NMG组[7.07%(3.40%),Z=-4.594,P<0.05]表达增高,CD14+单核细胞和CD19+B细胞上PD-L1+亚群较健康对照组及NMG组明显升高.MG患者外周血CD4+T上PD-1与ICOS的共表达[9.64%(8.82%)]较健康对照组[1.81%(2.10%),Z=-7.389,P<0.05]及NMG组[2.86%(1.49%),Z=-4.636,P<0.05]上调.(2)EILSA检测结果:MG组血清中sPD-1[(1.87 ±0.64) ng/ml]明显高于健康对照组[(1.49±0.70) ng/ml,t=2.04,P<0.05]及NMG组[(1.05±0.5) ng/ml,t=2.08,P<0.05];MG组血清中sPD-L1与健康对照组及NMG组差异无统计学意义.(3)血清细胞因子检测结果:MG组血清中IL-4表达[(61.88±5.15) pg/ml]较健康对照组[(32.03±1.84) pg/ml,t=2.50,P<0.05]及NMG组[(42.62±3.31) pg/ml,t=2.34,P<0.05]上调,MG患者sPD-1与IL-4呈正相关(r=0.406,P=0.029).结论 CD4+T细胞上的PD-1+ICOS+亚群升高提示该亚群参与了MG免疫病理过程,sPD-1可能干扰了T细胞上膜型PD-1的负性信号传导,引起ICOS正性信号相对亢进,导致该群细胞过度活跃推动MG病情进展.
更多Objective To explore the immunopathological mechanism for the imbalance between the positive signal mediated by inducible costimulator (ICOS) and the negative signal mediated by programmed death-1 (PD-1) in patients with myasthenia gravis (MG).Methods Eighty-two patients with MG,56 healthy controls (HC) and 20 non-MG (NMG) patients,collected in the First Affiliated Hospital of Suzhou University from February 2014 to December 2016,were chosen to participate in the study.The expression of ICOS and PD-1 on peripheral blood mononuclear cells was detected by immuno-fluorescence staining and flow cytometry.The levels of soluble programmed death-1 (sPD-1),soluble programmed death ligand 1 (sPD-L1),IL-4 and other cytokines were detected by enzyme-linked immunosorbent assay.Results (1) Flow cytometry analysis:The co-expression of PD-1,ICOS on CD4 + T cells from MG group (9.64% (8.82%)) was higher than in HC (1.81% (2.10%),Z =-7.389,P <0.05) and NMG group (2.86% (1.49%),Z =-4.636,P < 0.05).The expression of ICOS on CD4 + T cells,ICOS ligand (ICOSL) on CD14+ monocytes and CD19+ B cells were increased in MG group comparing with that of the control groups.The proportion of PD-1 + CD4 + T cells (MG group 16.82% (10.66%),HC 9.34% (9.18%),Z =-4.345,P<0.05;NMG group 7.07% (3.40%),Z=-4.594,P<0.05) and PD-1 Ligand (PD-L1) + CD14+ monocytes was higher in MG patients.All of these were detected by flow cytometry.(2) ELISA analysis:Serum sPD-1 expression significantly increased in MG group compared with that in the control groups (MG group (1.87 ± 0.64) ng/ml,NMG group (1.49 ± 0.70) ng/ml,t =2.04,P < 0.05;HC (1.05 ± 0.50)ng/ml,t =2.08,P < 0.05),while for serum sPD-L1,there was no significant difference between MG and control groups.(3) Serum cytokines detection:The expression of IL-4 was increased in MG patients (MG group (61.88 ±5.15) pg/ml,HC (32.03 ±1.84) pg/ml,t=2.50,P<0.05;NMG group (42.62± 3.31) pg/ml,t =2.34,P <0.05),and there was a negative correlation between the expression of sPD-1 and the concentration of IL-4.Conclusions The increased expression of PD-1 + ICOS + CD4 + T cells suggested the subset involved in the pathological progress of MG.sPD-1 might disturb the ligation of PD-1 on T cells and PD-L1 on antigen presenting cells,while the ligation of ICOS and ICOSL passed positive signal,leading to over activity of the subsets and the progression of disease.
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