IL-2和IL-7对优化检测Ⅰ型糖尿病患者谷氨酸脱羧酶65特异性T细胞反应的比较

摘要

Objective:To explore the type of cytokine (IL-2 or IL-7) and its most optimal concentration regarding the improvement of the signal-to-noise ratio of glutamic acid decarboxylase 65 (GAD65) in enzyme-linked immunospot (ELISPOT) assay in Type 1 diabetic (T1DM) patients.Methods:Twenty T1DM patients (Group A) and sixteen healthy controls matched with age and sex (Group B) were enrolled in our study,and their peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll method.GAD65,internal control and Pediacel served as "five-for-one" vaccine were selected as the stimulating antigen.Different concentrations of IL-2 [0 U/mL (Group 1),0.5 U/mL (Group 2),2.5 U/mL (Group 3) and 12.5 U/mL (Group 4)] were added to the culture system.The CD4+ T cells of secreting interferon-gamma (IFN-γ) in the above groups were determined by ELISPOT.The spots number,net values and stimulating index (SI) were compared in GAD65 (signal) and internal control (background).Next,another 21 T1DM patients (Group C) and 12 healthy controls matched with age and sex (Group D) were enrolled,and the specific T cell response to the GAD65 antigen was detected.The net values and SI were compared between the best optimal concentration of IL-2 (2.5 U/mL,Group 5) and IL-7 (0.5 ng/mL,Group 6).Results:1) After adding IL-2 into the Group A,the amount of GAD65 reactive T cells in different groups increased compared with Group A1,while the background in the internal control also increased gradually with the increased concentration of IL-2.There was no significant difference in net value (signal-noise) in the different concentration between the Group A3 and the Group A4 (P＞0.05).The SI in the Group A3 (2.8),the highest one,was significantly higher than that in the Group B3 (1.3) (P＜0.05).2) Although the number of GAD65 spots in the Group C6 and the Group D6 were slightly higher than that in the Group C5 and the Group D5,respectively,the background in the Group C6 and the Group D6 also increased,without statistical significance (P＞0.05).The mean net value spot and SI in the Group C5 (net value:5.5;SI:2.8) were both significantly higher than those in the Group C6 (net value:4.3;SI:1.8) (bothP9＜0.05).Conclusion:The concentration of 2.5 U/mL for IL-2 is proved to be the best optimal concentration for GAD65 specific T-cell responses in ELISPOT in patients with T1DM.IL-2 is much better than IL-7 in improvement of the SI in the ELISPOT.%目的:探讨何种细胞因子(IL-2或IL-7)及其最佳浓度能够最有效地改善酶联免疫斑点法(enzyme-linked immunospot,ELISPOT)检测1型糖尿病患者谷氨酸脱羧酶65(glutamic acid decarboxylase 65,GAD65)特异性T细胞反应的效率.方法:选取20例1型糖尿病患者(A组)及年龄和性别均匹配的16例正常对照者(B组);Ficoll法分离外周血单个核细胞(peripheral blood mononuclear cells,PBMCs),以人GAD65、内对照、巴斯德五合一疫苗为抗原,加入不同浓度的IL-2[0 U/mL(1组)、0.5 U/mL(2组)、2.5 U/mL(3组)和12.5U/mL(4组)],ELISPOT检测上述各浓度组分泌干扰素-γ(interferon-gamma,IFN-γ)的CD4+T细胞,比较各浓度组的GAD65孔(信号)、内对照孔(背景)的斑点数、斑点净值以及刺激指数(即信噪比,stimulating index,SI);另选取21例1型糖尿病患者(C组)及12例正常对照者(D组),检测针对GAD65抗原的特异性T细胞反应,比较联合IL-2(2.5 U/mL,上述实验已证明其为最优浓度,5组)与联合IL-7(0.5 ng/mL,6组)所产的斑点数和SI.结果:1)A且在加入IL-2刺激后,各浓度组中GAD65反应性T细胞数均较A1组增高,而各浓度下的内对照孔的斑点数亦呈比例升高.各浓度组斑点净值比较显示:A4组与A3组差异无统计学意义(P＞0.05),而SI的比较则提示A3组最高;且仅A3组的SI(2.8)高于B3组(1.5),差异有统计学意义(P＜0.05).2)C6组及D6组的GAD65孔斑点数均分别略高于C5组及D5组,且C6组及D6组的斑点数增幅也分别高于C5组及D5组,但差异均无统计学意义(P＞0.05).C5组的斑点净值(5.5)和SI(2.8)均显著高于C6组的斑点净值(4.3)和SI(1.8),差异均有统计学意义(均P＜0.05).结论:2.5 U/mL为ELISPOT检测1型糖尿病患者GAD65特异性T细胞反应的IL-2最佳浓度;IL-2较IL-7更有利于改善ELISPOT检测的SI.