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

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

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、χ^(2)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)×10^(6)/L、(97.11±17.10)×10^(6)/L比(43.86±9.52)×10^(6)/L,(57.15±10.58)×10^(6)/L、(82.27±15.15)×10^(6)/L比(42.35±9.33)×10^(6)/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。

Objective To determine the change of regulatory T cells in paroxysmal nocturnal hemoglobinuria(PNH)patients and analyze its probable immune mechanism in abnormal clones of PNH.Methods Thirty-one PNH patients admitted to Department of Hematology,Ningde Municipal Hospital of Ningde Normal University from January 2018 to January 2022 were selected as a case 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 healthy subjects were selected as a control group(14 males and 11 females,aged 19-56 years).Whole blood samples in subgroups were assayed by flow cytometer to detect T cells,Th lymphocyte subsets,and the numbers of CD4^(+)CD25^(+)regulatory T cells and Th3 cells.The expression levels of transforming growth factorβ(TGFβ)and forkhead box P3(FOXP3)were analyzed by real-time quantitative polymerase chain reaction(RT-PCR).Enzyme-linked immunosorbent assay(ELISA)kits were used to detect the plasma concentrations of TGF-βand interleukin-10(IL-10).F test,Bonferroni method,H test,and chi-square test were used.Results The number of CD4^(+)/CD3^(+)cells and the CD4^(+)/CD8^(+)ratio in the AA-PNH group were lower 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 episodic PNH 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 were higher than those in the control group[(67.82±15.67)×10^(6)/L,(97.11±17.10)×10^(6)/L vs.(43.86±9.52)×10^(6)/L;(57.15±10.58)×10^(6)/L,(82.27±15.15)×10^(6)/L vs.(42.35±9.33)×10^(6)/L],with statistically significant differences(all P<0.05).The positive expression rates of FOXP3 and TGF-βmRNA and the concentrations of TGF-βand IL-10 in the episodic PNH group and the AA-PNH group were higher than those in the 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/L vs.(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 and function of regulatory T cells in PNH patients are increased and its inhibitive function might contribute to abnormal clones of PNH to evade the immune surveillance.