T h17／T reg 细胞及相关细胞因子在复发缓解型多发性硬化中的作用及机制

摘要

目的 探讨辅助性T细胞(T h17) 、调节性 T细胞(T reg )和相关细胞因子在复发缓解型多发性硬化(RRM S)患者发病机制中的作用与机制.方法 收集急性期RRM S 患者(RRM S组)31例,以神经系统非炎性疾病29例为对照组. RRMS患者入院后给予静脉滴注甲泼尼龙1000 mg／d冲击治疗,后续每3 d剂量减半.对RRM S组患者治疗前和甲泼尼龙治疗2周后(治疗后)分别进行残疾状况拓展性量表(EDSS )评分;采用流式细胞术(FCM )检测 RRMS 组患者治疗前后及对照组患者外周血 T h17 (CD4+ IL-17+)细胞和 T reg (CD4+FOXP3+)细胞百分率;采用酶联免疫吸附试验(ELISA)检测RRM S组患者治疗前后及对照组患者外周血血浆中白细胞介素(IL )-17A 、IL-23 、IL-6 、IL-10 、IL-35和转化生长因子-β(TGF-β)水平;采用Spearman相关分析对RRM S组患者治疗前外周血 T h17细胞数量与 IL-17A 、IL-23 、IL-35 、IL-6 、IL-10及 TGF-β水平进行相关性分析.结果 (1)RRMS组治疗前EDSS评分高于治疗后〔分别(6.31 ± 1.54)分vs. (4.02 ± 0.68)分,t＝0.75 ,P＜0.05〕;(2)FCM 分析结果显示,与对照组〔(3.12 ± 1.27 )% 〕比较,RRM S组患者治疗前 T h17 细胞百分率〔(15.24 ± 2.54 )% 〕明显升高(P＜0.05) ,而对照组 T reg细胞百分率〔(35.04 ± 4.21 )% 〕明显高于RRM S组治疗前〔(11.12 ± 3.13 )%,P＜0.05〕;与对照组(0.10 ± 0.02 )相比,RRM S组治疗前T h17／T reg (1.51 ± 0.62)也明显升高(P＜0.01) ;与 RRM S组治疗前〔(11.12 ± 3.13 )% 〕比较,甲泼尼龙治疗后 T reg 细胞比例〔(23.14 ± 2.86)% 〕明显升高(P＜0.01) ;与RRM S组治疗前T h17细胞百分率〔(15.24 ± 2.54 )% 〕相比,甲泼尼龙治疗后Th17细胞百分率〔(4.24 ± 1.14)% 〕明显降低(t＝0.88 , P＜0.05) ;与RRMS组治疗前(1.51 ± 0.62 )相比,甲泼尼龙治疗后Th17／Treg 比值(0.19 ± 0.07 )降低(t＝0.95 , P＜0.01) ;(3)ELISA法检测结果显示,RRM S组治疗前IL-17A〔分别(17.26 ± 1.21 )pg／mL vs. (3.23 ± 0.81 )pg／mL ,t＝0.72 , P＜0.05〕、IL-23〔(分别(64.38 ± 7.51 ) pg／mL vs. (21.14 ± 1.82 )pg／mL ,t＝0.75 ,P＜0.05〕、IL-6〔分别(70.14 ± 8.17 )pg／mL vs. (7.28 ± 0.75 )pg／mL ,t＝0.95 ,P＜0.01〕和IL-10水平〔分别(21.12 ± 2.74 )pg／mL vs (2.39 ± 0.34 )pg／mL ,t＝0.91 ,P＜0.01〕均明显高于对照组,而 IL-35〔分别(0.31 ± 0.06 )pg／mL vs. (1.55 ± 0.16 )pg／mL ,t＝ －0.89 ,P＜ 0.01〕 和TGF-β水平〔分别(5.13 ± 0.34)pg vs. (18.25 ± 0.74)pg／mL ,t＝ －0.83 ,P＜0.05〕显著低于对照组;与RRM S组治疗前比较,治疗后IL-17A〔分别(17.26 ± 1.21 )pg／mL vs. (4.23 ± 0.90)pg／mL ,t＝0.82 ,P＜0.05〕、IL-23〔分别(64.38 ± 7.51 )pg／mL vs. (29.73 ± 2.51 )pg／mL ,t＝ 0.77 ,P＜0.05〕和 IL-6〔分别(70.14 ± 8.17 )vs. (15.23 ± 1.86 ) ,t ＝ 0.89 ,P ＜ 0.01〕水平明显降低,IL-35 (分别(0.31 ± 0.06 ) pg vs. (1.41 ± 0.18 ) pg , t＝ －0.88 ,P＜0.01)和TGF-β水平〔分别(5.13 ± 0.34)pg vs. (16.91 ± 0.59 )pg ,t＝ －0.72 ,P＜0.05〕明显升高;(4)RRM S组患者T h17细胞与 IL-17A (r＝ 0.89 ,P＜ 0.01 ) 、IL-23 (r＝ 0.71 ,P＜ 0.05 )和 IL-6 ( r＝ 0.83 , P＜0.05 )水平呈正相关,与IL-35和 TGF-β水平呈负相关〔分别 r＝ －0.82 ,P＜0.01 ;r＝ －0.74 ,P＜0.05〕.结论 RRM S的发生可能与 T h17细胞上调,T reg 细胞下调,T h17／T reg 表达失衡,相关细胞因子 IL-17A 、IL-23 、IL-6水平升高,IL-35和 TGF-β水平降低有着密切关系.%ABSTRACT : Objective To investigate the role of helper T cells (T h17) ,regulatory T cells (T reg ) and related cytokines in the pathogenesis of RRM S. Methods T hirty-one patients with acute RRM S relapses (the RRM S group) were collected ,and 29 patients with non-inflammatory neurological diseases were selected as the control group. After admission ,RRM S patients were treated with intravenous infusion of methylprednisolone at 1000 mg／d and the subsequent doses was halved every 3 days. Patients in the RRM S group were evaluated by the extended disability scale (EDSS) before treatment and after 2 weeks of treatment with methylprednisolone. Flow cytometry (FCM ) was used to detect the percentage of CD 4+ IL-17+ cells and CD4+ FOXP3+ cells in peripheral blood of the RRM S group before and after treatment as well as the control group. Serum levels of interleukin (IL-17A ,IL-23 , IL-6 , IL-10 , IL-35 and transforming grow th factor-β (TGF-β) in RRM S group and the control group were detected by ELISA before and after treatment. Spearman correlation analysis was used to analyze the correlation between the number of T h17 cells in peripheral blood and the levels of IL-17A , IL-23 ,IL-35 ,IL-6 , IL-10 and TGF-β. in the RRM S group before treatment. Results (1 ) The pre-treatment EDSS score in the RRM S group was higher than that after treatment [ (6. 31 ± 1. 54 ) vs. (4. 02 ± 0. 68) , t＝0. 75 , P＜0. 05 ] . (2) The results of the FCM analysis showed that compared with the control group [ (3. 12 ± 1. 27 )%] , the percentage of Th17 cells [ (15. 24 ± 2. 54)%] in the RRM S group before treatment was significantly increased ( P＜0. 05) ,w hile the percentage of T reg cells in the RRM S group before treatment was significantly lower [ (11. 12 ± 3. 13)% vs. (35. 04 ± 4. 21)%, P＜0. 01] . The Th17／T reg ratio of the RRMS group [ (1. 51 ± 0. 62 )%] also significantly increased before treatment ,compared with the control group [ (0. 10 ± 0. 02)%] ( P＜0. 01) . Compared with the RRM S group before treatment [ (11. 12 ± 3. 13)%] ,the percentage of T reg cells [ (23. 14 ± 2. 86 )%] after methylprednisolone treatment was significantly increased ( P＜0. 01) ,w hile the percentage of Th17 cells [ (15. 24 ± 2. 54)%] after methylprednisolone treatment was significantly decreased [ (4. 24 ± 1. 14)%] (t＝0. 88 , P＜0. 05) . Compared with the RRMS group before treatment [ (1. 51 ± 0. 62)%] ,Th17／Treg ratio was significantly reduced after treatment [ (0. 19 ± 0. 07 )%] (t＝ 0. 95 , P＜ 0. 01 ) . (4 ) The results of ELISA showed that IL-17A [ (17. 26 ± 1. 21 ) pg／mL vs. (3. 23 ± 0. 81 ) pg／mL , t＝ 0. 72 , P＜ 0. 05 ] , IL-23 [ (64. 38 ± 7. 51) pg／mL vs. (21. 14 ± 1. 82) pg／mL ,t＝0. 75 , P＜0. 05] ,IL-6 [ (70. 14 ± 8. 17) pg／mL vs. (7. 28 ± 0. 75) pg／mL ,t＝0.95 , P＜0. 01] ,and IL-10 [ (21. 12 ± 2. 74) pg／mL vs. (2. 39 ± 0. 34) pg／mL , t＝0. 91 , P＜0. 01] were significantly higher before RRM S treatment than the control group . While IL-35 levels [ (0. 31 ± 0. 06) pg／mL vs. (1. 55 ± 0. 16) pg／mL , t＝ －0. 89 , P＜0. 01] and TGF-β levels [ (5. 13 ± 0. 34) pg／mL vs. (18. 25 ± 0.74 ) pg／mL , t＝ －0. 83 , P＜ 0. 05 ] were significantly lower than those in the control group. Compared with the RRM S group before treatment ,IL-17A [ (17. 26 ± 1. 21) pg／mL vs. (4. 23 ± 0. 90 ) pg／mL ,t＝0.82 ,P＜ 0.05] ,IL-23 [ (64.38 ± 7.51) pg／mL vs. (29.73 ± 2.51) pg／mL ,t＝ 0.77 , P＜0. 05] ,and IL-6 [ (70. 14 ± 8. 17) pg／mL vs. (15. 23 ± 1. 86) pg／mL , t＝0. 89 , P＜0. 01 ] were significantly decreased after treatment. IL-35 [ (0. 31 ± 0. 06) pg／mL vs. (1. 41 ± 0. 18) pg／mL ,t＝ －0. 88 , P＜0. 01] and TGF-β levels [ (5. 13 ± 0. 34 ) pg／mL vs. (16. 91 ± 0. 59 ) pg／mL , t＝ －0. 72 , P＜ 0. 05 ] were significantly increased after treatment ( P ＜ 0. 05 , P ＜ 0. 01 ) . (4 ) In the RRM S group , Th17 cells were positively correlated with IL-17A (r＝0. 89 , P＜0. 01) ,IL-23 ( r＝0. 71 , P＜ 0. 05 ) and IL-6 ( r＝0. 83 , P＜ 0. 05 ) , while negatively correlated with IL-35 and TGF-β levels ( r ＝ 0. 82 , P ＜ 0. 01 , r ＝ － 0. 74 , P ＜ 0. 05 ). Conclusions The relapse of RRM S is closely related to the up-regulation of Th17 cells and down-regulation of T reg cells ,the imbalance of Th17／T reg expression ,the increase of IL-17A ,IL-23 and IL-6 ,and the decrease of IL-35 and TGF-β.