摘要目的:观察斑秃患者外周血白细胞介素35（IL-35）表达变化，评估IL-35对斑秃患者调节性T细胞（Treg）活性的调控。方法:收集2019年12月至2021年1月在山西省人民医院就诊的斑秃患者81例（斑秃组）和健康志愿者27例（对照组），分离血清和外周血单个核细胞（PBMC），酶联免疫吸附试验（ELISA）检测血清IL-35水平，实时荧光定量PCR法检测IL-35组成亚基EBI3和IL-12p35 mRNA表达，流式细胞仪检测CD4 +CD25 +CD127 dim/-Treg比例。使用重组人IL-35刺激纯化的Treg，ELISA检测培养上清液中穿孔素和颗粒酶B水平，实时荧光定量PCR检测EBI3、IL-12p35和免疫检查点分子程序性死亡蛋白1、黏蛋白结构域蛋白3、细胞毒性T淋巴细胞相关抗原4、淋巴细胞活化基因3 mRNA表达；将经IL-35刺激或未刺激的Treg与自体PBMC共培养，用细胞计数试剂盒8检测细胞增殖水平。计量资料两组之间比较采用 t检验，多组之间比较采用单因素方差分析，相关性分析采用Pearson检验；计数资料比较采用卡方检验。 结果:与对照组比较，斑秃组IL-35水平[（90.10 ± 11.98）ng/L比（100.74 ± 28.71）ng/L， t= 2.71， P= 0.008]、PBMC中EBI3 mRNA（1.06 ± 0.15比1.25 ± 0.11， t= 6.09， P < 0.001）、IL-12p35 mRNA（1.00 ± 0.15比1.38 ± 0.22， t= 10.16， P < 0.001）、Treg比例（5.91% ± 1.17%比6.85% ± 1.23%， t= 3.54， P= 0.001）均显著降低。斑秃组Treg比例与血清IL-35水平（ r= 0.25， P= 0.026）、PBMC中EBI3 mRNA（ r= 0.31， P= 0.004）、IL-12p35 mRNA水平（ r= 0.24， P= 0.032）均呈正相关。斑秃组未刺激的Treg分泌穿孔素、颗粒酶B水平以及EBI3、IL-12p35、免疫检查点分子mRNA表达均显著低于对照组Treg（ P < 0.05或0.001），抑制PBMC增殖的能力亦低于对照组（ P= 0.013）。重组人IL-35刺激后斑秃患者Treg分泌穿孔素和颗粒酶B水平与未刺激Treg相比无明显变化（ P > 0.05），但EBI3、IL-12p35、免疫检查点分子mRNA表达均显著升高（ P < 0.05或0.001），抑制PBMC增殖的能力亦增强（ P= 0.037）。 结论:斑秃患者外周血IL-35水平明显降低，与Treg功能减弱密切相关，可能参与斑秃发病。

abstractsObjective:To investigate changes in the peripheral interleukin-35 (IL-35) level in patients with alopecia areata, and to assess its modulatory effect on regulatory T (Treg) cell activities.Methods:Totally, 81 patients with alopecia areata (alopecia areata group) and 27 healthy volunteers (control group) were enrolled from Shanxi Provincial People′s Hospital between December 2019 and January 2021. Sera and peripheral blood mononuclear cells (PBMCs) were isolated. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the serum IL-35 level, real-time fluorescence-based quantitative PCR to determine the mRNA expression of IL-35 subunits EBI3 and IL-12p35, and flow cytometry to determine the proportion of CD4 + CD25 + CD127 dim/- Treg cells. Sorted Treg cells were stimulated by recombinant human IL-35, ELISA was performed to detect levels of perforin and granzyme B in the culture supernatant, and real-time fluorescence-based quantitative PCR to determine the mRNA expression of EBI3, IL-12p35, and immune checkpoint molecules, such as programmed death protein 1 (PD-1) , T cell immunoglobulin and mucin protein-3 (Tim-3) , cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and lymphocyte activation gene-3 (LAG-3) in Treg cells. IL-35-stimulated or unstimulated Treg cells were co-cultured with autologous PBMCs, and cell counting kit-8 (CCK8) assay was conducted to assess cellular proliferative activity. Measurement data were compared between 2 groups by using t test, comparisons among multiple groups were carried out by using one-way analysis of variance, correlation analysis was carried out by using Pearson correlation analysis, and enumeration data were compared by using chi-square test. Results:Compared with the control group, the alopecia areata group showed significantly decreased IL-35 levels (90.10 ± 11.98 ng/L vs. 100.74 ± 28.71 ng/L, t= 2.71, P= 0.008) , mRNA expression of EBI3 and IL-12p35 in PBMCs (EBI3: 1.06 ± 0.15 vs. 1.25 ± 0.11, t= 6.09, P < 0.001; IL-12p35: 1.00 ± 0.15 vs. 1.38 ± 0.22, t= 10.16, P < 0.001) , and proportions of Treg cells (5.91% ± 1.17% vs. 6.85% ± 1.23%, t= 3.54, P= 0.001) . In the alopecia areata group, the proportion of Treg cells was positively correlated with the serum IL-35 level ( r= 0.25, P= 0.026) , and the mRNA expression of EBI3 and IL-12p35 in PBMCs ( r= 0.31, 0.24, P= 0.004, 0.032, respectively) . Compared with the control group, the unstimulated Treg cells from the alopecia areata group showed significantly decreased supernatant levels of perforin and granzyme B, mRNA expression of EBI3, IL-12p35 and immune checkpoint molecules ( P < 0.05 or 0.001) , as well as weakened inhibitory effect on the proliferative activity of PBMCs ( P= 0.013) . There was no significant difference in the level of perforin or granzyme B between the recombinant human IL-35-stimulated and unstimulated Treg cells from the patients with alopecia areata (both P > 0.05) . However, the mRNA expression of EBI3, IL-12p35 and immune checkpoint molecules was significantly higher in the IL-35-stimulated Treg cells than in the unstimulated Treg cells in the alopecia areata group ( P < 0.05 or 0.001) , and the inhibitory effect on the proliferative activity of PBMCs was also significantly enhanced in the IL-35-stimulated Treg cells compared with the unstimulated Treg cells ( P= 0.037) . Conclusion:The peripheral IL-35 level was significantly decreased in the patients with alopecia areata, which was closely associated with reduced activities of Treg cells, and IL-35 may be involved in the occurrence of alopecia areata.