CD4+CD25+调节性T细胞在自身免疫性甲状腺疾病中数量和功能变化

摘要

目的:探讨自身免疫性甲状腺疾病患者外周血中CD4+CD25+调节性T细胞(Tregs)的数量和功能变化.方法:采用化学发光法测定20例初发Graves'病人、20例初发桥本甲状腺炎(HT)患者及20例健康体检者血清中促甲状腺素(TSH)、总三碘甲状腺原氨酸(TT3)、总甲状腺素(TT4)、甲状腺球蛋白抗体(TgAb)和甲状腺过氧化酶抗体(TPOAb)的水平;用流式细胞仪分析外周血单个核细胞(PBMC)中CD4+T细胞及CD4+CD25+ Tregs的数量;采用磁珠分选技术分选5例HT病人和5例健康体检者PBMC中CD4+CD25+ Tregs和CD4+CD25-T细胞,采用MTT法检测CD4+CD25+ Tregs对自身CD4+CD25-T细胞增殖的抑制作用;提取各组PBMC的总RNA,经Real time-PCR检测TGF-β1、Foxp3 mRNA的表达水平.结果:流式细胞检测结果显示,初发Graves'病人、初发HT患者外周血PBMC中CD4+T细胞数量与正常人比较无差异(P＜0.05);初发HT患者外周血PBMC中CD4+CD25+ Tregs占CD4+T细胞的比率为(1.55%±0.49%),明显低于正常对照组(2.86%±1.04%)(P＜0.05);初发Graves'病人外周血PBMC中CD4+CD25+ Tregs占CD4+T细胞的比率为(3.25%±0.97%),与正常对照组(2.86%±1.04%)相比无显著性差异(P＜0.05).MTT结果显示,初发HT患者CD4+CD25+ Tregs对自身CD4+CD25-T细胞增殖的抑制百分率为15.7%±5.36%,与正常组(41.7%±9.87%)相比显著降低(P＜0.05).Real time-PCR结果显示,初发Graves'病人、初发HT患者PBMC的TGF-β1 mRNA表达水平分别为(0.37±0.10)和(0.43±0.09),均明显低于正常对照组(1.02±0.04)(P＜0.05);初发Graves'病人、初发HT患者PBMC的Foxp3 mRNA表达水平分别为0.62±0.09和0.42±0.29,均明显低于正常对照组(0.99±0.17)(P＜0.05).结论:本研究结果提示,HT患者外周血中CD4+CD25+ Tregs的数量和功能明显降低.Graves'病和HT患者外周血PBMC中TGF-β1、Foxp3 mRNA表达水平明显降低.%Objective :The aim of this study is to explore the frequency and the function of circulating CD 4+ CD25+ regulatory T cells (Tregs ) in the patients with autoimmune thyroid diseases (AITD ) .Methods :The serum levels of thyroid stimulating honnone (TSH ) ,total triiodothyronine (TT3 ) ,total thyroxine (TT4 ) , thyroglobulin antibody (TgAb ) and thyroid pemxidase antihody (TPOAb ) were measured with commercial electrochemiluminescence (ECL) Kits .20 patients of Graves ' disease (GD ) patients and 20 Hashimoto ' s thyroiditis (HT ) were enrolled ,with the samples of 20 healthy control subjects .Peripheral blood mononuclear cells (PBMC ) were freshly isolated form all the groups . The proportion of circulating CD4+ CD25+ Tregs was assessed by flow cytometry .CD4+ CD25+ Tregs were isolated from five HT patients and five healthy control subjects using Magnetic Activated Cells Sorting ,and the suppressive function of CD4+ CD25+ Tregs on autologous CD4+ CD25 - T cells were determined by MTT method .The mRNA expressing levels of TGFβ1 and Foxp3 of PBMC were detected by real-time quantitative PCR .Results :A significantly decreased proportion of the circulating CD4+ CD25+ Tregs was observed in HT patients (1 .55％＜ 0.49％ vs 2 .86％ ±1 .04％ , P＜0 .05 ) .There was a diminished suppression of CD4+ CD25+ Tregs from HT patients on autologous CD4+ CD25- responder T cell proliferation when compared with controls (15 .7％ ±5 .36％ vs 41 .7％ ±9 .87％ , P＜ 0 .05 ) .Lower mRNA expres sion of TGF-β1 and Foxp3 was observed both in GD and HT patients compared to control subjects ( P＜ 0 .05 ) .Conclusion :Our results indicate that there is a decreased proportion of CD4+ CD25+ Tregs which are functionally defective in HT patients .The mRNA expressing levels of TGF β1 and Foxp3 are reduced in GD patients and HT patients .