自身免疫性溶血性贫血／Evans综合征患者外周血CD4^＋CD69^＋T细胞的临床意义

The clinical significance of CD4^＋CD69^＋ T lymphocytes in patients with autoimmune hemolytic anemial Evans syndrome

目的 了解自身免疫性溶血性贫血（AIHA）/Evans综合征（ES）患者外周血CD4^＋CD69^＋T细胞的临床意义.方法 选取2015年10月至2016年5月于天津医科大学总医院血液科住院的AIHA/ES患者32例,其中溶血发作组15例,缓解组17例;健康对照13名.以流式细胞仪检测外周血CD69^＋T细胞;实时定量PCR检测外周血CD4^＋T细胞中CD69 mRNA表达水平;酶联免疫吸附试验（ELISA）检测血浆CD69浓度.结果 （1）溶血发作组的CD3^＋CD69^＋/CD3^＋T细胞比例高于缓解组[（3.08±1.48）％比（1.96±1.33）％,P＜0.05]和健康对照组[（1.28±0.83）％,P＜0.01];溶血发作组CD3^＋CD69^＋T细胞绝对值高于健康对照组[（2.94±1.81）× 10^7/L比（1.48± 1.42）× 10^7/L,P＜0.05].（2）溶血发作组CD3^＋CD4^＋CD69^＋/CD3^＋CD4^＋T细胞比例高于缓解组[（2.16±1.56）％比（1.16±0.62）％,P＜0.05]及健康对照组[（0.94±0.78）％,P＜0.05];溶血发作组CD3^＋CD4^＋CD69^＋T细胞绝对值高于健康对照组[（1.04±0.98）× 10^7/L比（0.44±0.38）× 10^7/L,P＜0.05].（3）溶血发作组CD3^＋CDs＋CD69^＋/CD3^＋CD8^＋T细胞比例高于健康对照组[（4.87±2.56）％比（1.83±1.27）％,P＜0.01];但CD3^＋CD8^＋CD69^＋T细胞绝对值在3组间差异无统计学意义.（4） Pearson相关分析显示,溶血发作组CD3^＋CD4^＋CD69^＋/CD3^＋CD4^＋T细胞比例与血红蛋白浓度呈负相关（P.＜0.01）,与总胆红素以及间接胆红素均呈正相关（P均＜0.01）,与网织红细胞比例呈正相关（P=0.01）;缓解组CD3^＋CD4^＋CD69^＋/CD3^＋CD4^＋T细胞比例与血红蛋白浓度呈负相关（P＜0.05）.（5）溶血发作组CD4^＋T细胞中CD69mRNA的相对表达水平显著高于缓解组（32.26±35.11比6.05±5.87,P＜0.05）及健康对照组（1.76±1.85,P＜0.01）,缓解组高于健康对照组（P＜0.05）.（6）溶血发作组患者血浆CD69水平高于健康对照组[（494.21±16.06） ng/L比（441.39±104.6） ng/L,P＜0.05].结论 AIHA/ES患者外周血CD69^＋T细胞比例增高,以CD4^＋CD69^＋T细胞增高为主,且与AIHA/ES的疾病严重程度相关.

Objective To investigate the significant of peripheral CD4^＋CD69^＋T lymphocytes in patients with autoimmune hemolytic anemia （AIHA）/Evans syndrome （ES）.Methods In this study peripheral blood samples from 32 patients with AIHA/ES （15 hemolytic episode patients,17 remission patients） and 13 healthy controls were collected.Patients with AIHA/ES were recruited in Tianjin Medical University General Hospital from October 2015 to May 2016.The percentages of CD69^＋ T lymphocytes were analyzed by flow cytometry.The expression of CD69 mRNA in CD4^＋T lymphocytes which was sorted from peripheral blood by magnetic activated cell sorting （MACS） was detected using real-time PCR.Soluable CD69 was measured by ELISA.Results In hemolytic episode patients,the ratio of CD3^＋CD69^＋/CD3^＋T lymphocytes [（3.08 ± 1.48）%] was significantly higher than that in healthy controls [（1.28 ± 0.83）%,P〈0.01] and in remission group[（1.96± 1.33）%,P〈0.05].The absolute count of CD3^＋CD69^＋T lymphocytes in hemolytic episode group [（2.94± 1.81）× 10^7/L] was higher than that in healthy controls [（1.48± 1.42）× 10^7/L,P〈0.05].The ratio of CD3^＋CD4^＋CD69^＋/CD3^＋CD4^＋T cells in hemolytic episode group [（2.16± 1.56）%] was significantly higher than that in remission group [（1.16±0.62）%,P〈0.05] and healthy controls[（0.94±0.78）%,P〈0.05].The quantity of CD3^＋CD4^＋CD69^＋T lymphocytes in hemolytic episode group[（1.04±0.98）× 10^7/L] was higher than in healthy controls [（0.44± 0.38） × 10^7 / L,P〈0.05].The ratio of CD3^＋CD8^＋CD69^＋/CD3^＋ CD8^＋T lymphocyte in hemolytic episode group [（4.87±2.56）%] was significantly higher than that in healthy controls[（1.83± 1.27）%,P〈0.01].The quantity of CD3^＋CDs＋CD69^＋T lymphocytes in three groups did not show significant difference.The ratio of CD3^＋CD4^＋CD69^＋/CD3^＋CD4^＋T lymphocytes in hemolytic episode group was negatively correlated with hemoglobin （Hb） （P〈0.01）,positively correlated with the percentage of reticulocytes （Ret%）（P=0.01）total bilirubin（TBil）,indirect bilirubin（IBil） （P〈0.01） and not correlated with absolute reticulocytes count,lactic dehydrogenase （LDH）,complement 3（C3）,complement 4 （C4）.The ratio of CD3^＋CD4^＋CD69^＋/CD3^＋CD4^＋T lymphocytes in remission group was negatively correlated with Hb （P〈0.05）.In hemolytic episode patients CD69 mRNA （32.26±35.11） was significantly higher than that in remission group（6.05±5.87）（P〈0.05）and healthy controls （1.76± 1.85）（P〈0.01）.CD69 mRNA in remission group was significantly higher than healthy controls （P〈0.05）.Serum CD69 in hemolytic episode patients [（494.21 ± 16.06） ng/L] was significantly higher than that in healthy controls [（441.39± 104.6） ng/L,P〈0.05].Conclusion Our findings suggest that the proportion of CD4^＋CD69^＋ T lymphocytes increase in AIHA/ES patients,which is correlated with the severity of disease.