Th17/Treg细胞失衡在儿童再生障碍性贫血免疫发病及治疗中的意义

Role of Th17/Treg imbalance in the immune pathogenesis and treatment of children with aplastic anemia

目的 探讨Th17/Treg细胞失衡在儿童再生障碍性贫血（AA）免疫发病及治疗过程中的意义.方法 选取2012年1月至2013年10月在安徽医科大学第二附属医院收治的AA患儿43例（其中男14例）,年龄2～ 14岁,根据AA患儿初诊时疾病严重程度分为2组：重型AA（SAA,25例,其中男8例,年龄2 ～14岁）和非重型AA（NSAA,18例,其中男6例,年龄2～14岁）.随着治疗的进行,根据临床实际情况制定3个监测时间点：初诊（43例,其中男14例,年龄2～14岁）,脱离输血（8例,其中男5例,年龄2 ～11岁）和完全缓解（6例,其中男3例,年龄2～11岁）,以同期治疗无效的AA患儿作为无效对照（脱离输血无效对照,5例,其中男1例,年龄3～8岁;完全缓解无效对照,4例,其中男2例,年龄4～11岁）.采用流式细胞术检测各研究对象外周血中Treg及Th17细胞占CD4＋T细胞的比例,并计算Th 17/Treg;酶联免疫吸附试验（ELISA法）检测初诊AA患儿血浆中白细胞介素（IL）-17和IL-6水平.并检测本院同期25名（其中男12名,年龄3～ 14岁）健康体检儿童外周血中Treg及Th17细胞比例及血浆中IL-17和IL-6水平作为健康对照,以同期9例（其中男4例,年龄1～11岁）化疗后AA状态患儿各项指标作为初诊对照组.采用t检验或ANOVA分析及Mann-Whitney U或Kruskal-WallisH检验进行组间比较;相关性分析采用Spearman相关系数分析.结果 （1）初诊AA患儿外周血中Th17细胞比例1.63％（1.27％,2.48％）高于健康对照组0.40％ （0.35％,0.51％）和初诊对照组0.50％ （0.45％,0.75％）;Treg细胞比例4.24％ （3.10％,5.29％）低于正常对照组7.03％ （6.56％,7.48％）和初诊对照组7.50％（6.60％,8.30％）;同时Th17/Treg 0.53 （0.34,0.69）高于正常对照组0.06（0.05,0.07）和初诊对照组0.09（0.08,0.11）;差异均有统计学意义（P均＜0.01）.（2）初诊AA患儿血浆中的IL-17和IL-6均高于正常对照组[（223 ±92）比（116±18）ng/L,（26.2±12.0）比（10.6±2.1）ng/L,P均＜0.01].初诊AA患儿IL-17和IL-6水平均与Th17细胞比例成正相关（r =0.62、0.64,P均＜0.01）.（3）SAA患儿外周血中Th17细胞比例、Th17/Treg、IL-17及IL-6均高于正常对照组[1.80％（1.25％,2.61％）比0.40％ （0.35％,0.51％）,0.57％ （5.10％,0.82％）比0.06％ （0.05％,0.07％）,（225±108）比（116±18） ng/L,（25.9±12.6）比（10.6±2.1）ng/L,P均＜0.01];NSAA患儿外周血中Th17细胞比例、Th17/Treg、IL-17及IL-6均高于正常对照组,但SAA及NSAA患儿的Treg细胞比例均低于正常对照组（P均＜0.01）.SAA与NSAA患儿之间的Th17及Treg细胞比例、Th 17/Treg、IL-17及IL-6水平差异均无统计学意义（P均＞0.05）.（4）不同治疗阶段（初诊、脱离输血、完全缓解、脱离输血无效对照、完全缓解无效对照）AA患儿Th17细胞比例和Th 17/Treg差异均有统计学意义（P均＜0.05）,Treg比例差异均无统计学意义（P均＞0.05）.结论 AA患儿外周血中存在Th 17/Treg细胞失衡及TH17细胞相关因子异常增多现象,但对初诊AA的严重程度无明显影响.经免疫抑制治疗后,随着Th17/Treg失衡的纠正,AA得到逐步缓解.

Objective To study the role of Th17/Treg imbalance in the immune pathogenesis and therapeutic significance in childhood aplastic anemia （AA）.Method We analyzed data from 43 children （male∶female =14∶ 29） with AA,all the cases were at the age of 2 to 14 years at diagnosis,and were hospitalized at our department of pediatrics between January 2012 and October 2013 in the Second Hospital of Anhui Medical University.All these patients were divided into 2 groups,severe AA （SAA） group （n =25,male∶ female =8∶ 17,2-14 years old） and non-severe AA （NSAA） group （n =18,male∶ female =6∶ 12,2-14 years old）,depending on the severity at first diagnosis.As to the treatment,we analyzed data at 3 phases of treatment,diagnosis （n =43,male ∶ female =14 ∶ 29,2-14 years old）,transfusionindenpendence （n =8,male∶ female =5∶3,2-11 years old）,complete response （n =6,male∶ female =3∶3,2-11 years old）; at the same time,AA children who did not respond to the treatments were considered as failed treatment control （transfusion-indenpendence with failed treatment group,n =5,male∶ female =1∶4,3-8 years old; complete response failed treatment group,n =4,male∶ female =2∶ 2,4-11 years old）.The ratio of Treg and Th17 cells in CD4＋ T cells were tested by flow cytometry.The levels of IL-6 and IL-17 in plasma were determined by ELISA.During the same period,25 age-matched healthy children （male∶ female =12∶ 13,3-14 years old） were recruited as normal control,9 cases （male∶ female =5∶3,2-11 years old） of AA children induced by chemotherapy as diagnosis control group.Differences in variables were analyzed using ANOVA and t-tests or the Kruskal-Wallis and Mann-Whitney Utests,as appropriate.Correlation analysis was evaluated by the Spearman rank correlation test.Result （1） The ratio of Th17 cells in newly diagnosed AA patients were higher than that of normal group or diagnosis control group [1.63％（1.27％,2.48％） vs.0.4％ （0.35％,0.51％） or 0.50％ （0.45％,0.75％）,both P ＜0.01] while the ratio of Treg cells was lower [4.24％ （3.10％,5.29％） vs.7.03％ （6.56％,7.48％） or 7.50％ （6.60％,8.30％）,both P ＜0.01] and the proportion of Th17/Treg were significantly higher [0.53 （0.34,0.69） vs.0.06 （0.05,0.07） or 0.09 （0.08,0.11）,both P ＜ 0.01].（2） The levels of IL-6 and IL-17 in newly diagnosed AA patients were higher than in normal group [（223 ± 92） vs.（116 ± 18） ng/L,（26.2 ± 12.0） ng/L vs.（10.6 ±2.1） ng/L,P both ＜0.01].There was a positive correlation between Th17 cells and some Th17 cells related cytokines such as IL-17 and IL-6 （r =0.62,0.64,P both ＜0.01）.（3） The ratio of Th17,Th17/Treg,and the levels of IL-6 and IL-17 in children with SAA were also higher than in normal group [1.80％ （1.25％,2.61％） vs.0.40％ （0.35％,0.51％）,0.57％（5.10％,0.82％） vs.0.06％ （0.05％,0.07％）,（225±108） vs.（116±18） ng/L,（25.9 ± 12.6） vs.（10.6 ±2.1）ng/L,all P ＜0.01].NSAA also higher than normal group.The ratio of Treg in children with SAA and NSAA was less than that in normal group （P all ＜0.01）.However,the ratio of Th17,Treg,Th17/Treg,and the levels of IL-6 and IL-17 had no significant difference between SAA and NSAA （all P ＞ 0.05）.（4） In different stages of treatment,such as diagnosis,transfusion-indenpendence,complete response,there were significant differences in the ratio of Th17 and Th17/Treg （both P ＜ 0.05） but not in Treg （P ＞ 0.05）.Conclusion The imbalance of Th17/Treg cells and abnormally increased cytokines related to Th17 cells exist in peripheral blood of AA children,but did not significantly affect the severity of AA in preliminary diagnosis.After treatment with immunosuppression,AA was gradually relieved as the imbalance of Th17/Treg was corrected.