调节性T细胞在阵发性睡眠性血红蛋白尿症异常克隆中的免疫机制研究

目的　测定阵发性睡眠性血红蛋白尿症（PNH）患者调节性T细胞的变化，探讨其在PNH异常克隆中可能的免疫机制。方法　2018年1月至2022年1月在宁德师范学院附属宁德市医院血液科就诊的31例PNH患者为病例组，其中发作性PNH 15例（男10例、女5例，年龄23～69岁）、再生障碍性贫血-阵发性睡眠性血红蛋白尿（AA-PNH）16例（男11例、女5例，年龄20～68岁）；25例健康体检者为对照组（男14例、女11例，年龄19～56岁）。用流式细胞仪检测各组外周血T细胞和Th细胞亚群变化以及CD4⁺CD25⁺T细胞和Th3细胞数量，用实时荧光定量多聚核苷酸链式反应（RT-PCR）测定转化生长因子-β（TGF-β）和叉头框蛋白P3（FOXP3）的表达水平，用酶联免疫吸附试验（ELISA）检测TGF-β和白细胞介素-10（IL-10）浓度。采用F检验、Bonferroni方法、H检验、χ²检验。结果　AA-PNH组CD4⁺/CD3⁺细胞和CD4⁺/CD8⁺比例低于对照组［（44.55±11.91）%比（57.61±6.90）%、0.66（0.38，1.26）比1.32（0.86，1.64）］，CD8⁺/CD3⁺细胞高于对照组［（62.31±10.76）%比（45.51±5.54）%］，差异均有统计学意义（均P<0.05）；AA-PNH组Th1/CD3⁺CD4⁺细胞高于发作性PNH组及对照组［（24.27±4.30）%比（12.61±3.02）%、（4.07±1.93）%］，差异有统计学意义（P<0.05）。AA-PNH组及发作性PNH组CD4⁺CD25⁺ T细胞和Th3细胞的数量高于对照组［（67.82±15.67）×10⁶/L、（97.11±17.10）×10⁶/L比（43.86±9.52）×10⁶/L，（57.15±10.58）×10⁶/L、（82.27±15.15）×10⁶/L比（42.35±9.33）×10⁶/L］，差异均有统计学意义（均P<0.05）。发作性PNH组、AA-PNH组中FOXP3及TGF-β的mRNA的阳性表达率和TGF-β及IL-10浓度均高于对照组［86.67%（13/15）、75.00%（12/16）比40.00%（10/25），73.33%（11/15）、62.50%（10/16）比36.00%（9/25），（49.76±2.03）μg/L、（37.29±1.78）μg/L比（17.01±3.27）μg/L，（7.86±1.17）pg/ml、（5.57±1.13）pg/ml比（1.33±0.49）pg/ml］，差异均有统计学意义（均P<0.05）。结论　PNH患者调节性T细胞数量增加，功能亢进，推测其免疫抑制作用可能使其在PNH异常克隆中逃避免疫监视。

Study of immune mechanism of regulatory T cells in abnormal clones ofparoxysmal nocturnal hemoglobinuria

Objective　To determine the change of regulatory T cells in paroxysmal nocturnalhemoglobinuria (PNH) patients and analyze its probable immune mechanism inabnormal clones of PNH. Methods　Thirty-one PNHpatients admitted to Department of Hematology, Ningde Municipal Hospital ofNingde Normal University from January 2018 to January 2022 were selected as acase group, including fifteen cases of episodic PNH (10 males and 5 females,aged 23-69 years) and sixteen cases of aplastic anemia-paroxysmal nocturnal hemoglobinuria(AA-PNH) (11 males and 5 females, aged 20-68 years). Twenty-five healthysubjects were selected as a control group (14 males and 11 females, aged 19-56years). Whole blood samples in subgroups were assayed by flow cytometer todetect T cells, Th lymphocyte subsets, and the numbers of CD4⁺CD25⁺ regulatory T cells and Th3 cells. The expression levels of transforming growthfactor-β (TGF-β) and forkhead box P3 (FOXP3) were analyzed by real-timequantitative polymerase chain reaction (RT-PCR). Enzyme-linked immunosorbentassay (ELISA) kits were used to detect the plasma concentrations of TGF-β andinterleukin-10 (IL-10). F test,Bonferroni method, H test, andchi-square test were used. Results　The number of CD4⁺/CD3⁺ cells and the CD4⁺/CD8⁺ ratio in the AA-PNH group werelower than those in the control group [(44.55±11.91)% vs. (57.61±6.90)%, 0.66(0.38, 1.26) vs. 1.32 (0.86, 1.64)], but the CD8⁺/CD3⁺ cells were higher than those in the control group [(62.31±10.76)% vs.(45.51±5.54)%], with statistically significant differences (all P<0.05). The number of Th1/CD3⁺CD4⁺ cells in the AA-PNH group was significantly higher than those in the episodicPNH group and the control group [(24.27±4.30)% vs. (12.61±3.02)%,(4.07±1.93)%], with a statistically significant difference (P<0.05). The numbers of CD4⁺CD25⁺ T cells and Th3 cells in the AA-PNH group and the episodic PNH group werehigher than those in the control group [(67.82±15.67) ×10⁶/L,(97.11±17.10) ×10⁶/L vs. (43.86±9.52) ×10⁶/L;(57.15±10.58) ×10⁶/L, (82.27±15.15) ×10⁶/L vs.(42.35±9.33) ×10⁶/L], with statistically significant differences(all P<0.05). The positiveexpression rates of FOXP3 and TGF-β mRNA and the concentrations of TGF-β andIL-10 in the episodic PNH group and the AA-PNH group were higher than those inthe control group [86.67% (13/15), 75.00% (12/16) vs. 40.00% (10/25); 73.33%(11/15), 62.50% (10/16) vs. 36.00% (9/25); (49.76±2.03) μg/L, (37.29±1.78) μg/Lvs. (17.01±3.27) μg/L; (7.86±1.17) pg/ml, (5.57±1.13) pg/ml vs. (1.33±0.49)pg/ml], with statistically significant differences (all P<0.05). Conclusion　The number andfunction of regulatory T cells in PNH patients are increased and its inhibitivefunction might contribute to abnormal clones of PNH to evade the immunesurveillance.