Graves病患者外周血CD4^+、CD25^+调节性T细胞的定量及功能分析

Quantitation and functional analysis of CD4^+,CD25^+ T regulatory cells in patients with Graves disease

目的:检测Graves病患者外周血CD4+、CD25+免疫调节性T细胞(Tregulatory cells,Tregs)数量并对其进行免疫抑制功能分析,探讨Tregs细胞在其发病机制中的作用。方法:利用不同荧光标记的鼠抗人CD3、CD4、CD25单克隆抗体标记患者及对照组外周血淋巴细胞,流式细胞仪定量检测Tregs细胞占CD4+T细胞的百分比。用CD4细胞阴性选择磁珠分离CD4细胞,用抗CD25的磁珠分选CD25+Tregs和CD25-Tregs的效应性T细胞(T responder cells,Treps),用体外细胞培养和3H-thymidine掺入法测定Tregs细胞对Treps细胞的免疫抑制功能。结果:36例Graves病患者与20例健康对照组外周血CD4+T细胞中Tregs细胞分别为(6.96±1.98)%及(8.22±2.78)%,两者差异无统计学意义(P>0.01)。Graves病患者的Tregs细胞可以抑制自身Treps细胞的体外增殖,1∶1、1∶2、1∶4、0∶1比例平均抑制率分别为(48.2±4.9)%、(29.6±7.1)%、(17.7±5.6)%、0,健康对照组的平均抑制率分别为(73.1±5.3)%、(51.5±5.8)%、(29.6±6.3)%、0。Graves病组与健康对照组相比平均抑制率降低,差异有统计学意义(P<0.01)。结论:Graves病患者外周血中Tregs细胞的数量与对照组没有差异,但其免疫抑制功能明显降低,提示Tregs细胞功能的降低与Graves病的致病机制存在关联。

Objective： Percentage of CD4^＋ CD25^＋ T regulatory cells （Tregs） on peripheral blood was measured in patients with Graves disease and suppression function of Tregs cells were analyzed in vitro to study the function of Tregs cells in pathogenesis of Graves disease. Methods： Three - colour flow- cytometry analysis of Tregs was performed on fresh peripheral blood in patients with Graves disease and in control group. CD4 cell was isolated by CD4 positive selection using immunomagnetic beads and CD25^＋ Tregs cells and T responder cells （Treps） of CD25^- were isolated by CD25 positive selection using immunomagnetic beads. The isolated cell were cultured and suppression functions were detected by ^3H- thymidine experiment. Results： The percentages of Tregs in Graves disease patients and in control group were （6.96 ± 1.98） % and （8.22 ± 2.78） %, respectively, between the difference of which there was no statistical significance （P 〉0.01 ）. The Tregs in patients with Graves disease could suppress increasement of self- Treps in vitro. When the ratios of Tregs and Treps were 1 ： 1, 1：2, 1 ： 4, 0： 1, the inhibition ratio on average were （48.2 ± 4.9） %, （29.6 ±7.1）%, （17.7±5.6）% and zero, respectively. Whereas those in control group were （73.1±5.3）%, （51.5±5.8）%, （29.6 ± 6.3） % and zero, respectively. There were statistical differences between both groups （P 〈 0.01 ）. Conclusions： The patients with Graves disease have a percentage of Tregs same as the normal controls, but suppression function of Tregs in Graves disease patients significantly decrease, which indicate that the change of function of Tregs may play an important role in the pathogenesis of Graves disease.