初发1型糖尿病患儿外周血单个核细胞FOXP3和CTLA-4表达的研究

The expression of FOXP3 and CTLA-4 in peripheral blood mononuclear cells in children with newly onset type 1 diabetes mellitus

目的：研究初发1型糖尿病患儿外周血叉状头转录因子（ FOXP3）和细胞毒性T细胞相关抗原-4（CTLA-4）表达水平,探讨它们在1型糖尿病发病中的作用。方法选取50例初发1型糖尿病患儿和30例健康儿童,采用real-time PCR法研究FOXP3和CTLA-4 mRNA表达；ELISA方法检测血清中可溶性FOXP3（ sFOXP3）和CTLA-4（ sCTLA-4）蛋白水平；分别应用免疫印记法、高效液相离子层析法和电化学发光法测量糖尿病抗体、HbA1C及C肽。结果1型糖尿病患儿FOXP3 mRNA及蛋白表达低于对照组[0.95±0.48 vs.2.11±0.79,（6.27±1.49） ng/ml vs.（9.02±2.37） ng/ml,均P〈0.01],而CTLA-4 mRNA及蛋白表达高于对照组[2.43±0.83 vs.1.94±0.84,（77.88±22.34） ng/ml vs.（65.97±12.11） ng/ml,P〈0.01]；1型糖尿病患儿FOXP3和CTLA-4基因与蛋白表达均呈正相关（r＝0.758、0.396,均P〈0.05）；FOXP3与CTLA-4蛋白表达具有相关性（r＝－0.624,P〈0.05）。结论初发1型糖尿病患儿外周血FOXP3和CTLA-4的基因及蛋白表达异常,FOXP3调控CTLA-4在调节性T 细胞的表达,提示免疫机制参与1型糖尿病的发生。

Objective To investigate the expression levels of forkhead transcription factor 3 （FOXP3） and cytotoxic T lymphocyte-associated antigen-4（CTLA-4） in the peripheral blood of children with newly diagnosed type 1 diabetes mellitus（T1DM）,and explore their roles in the pathogenesis of T1DM. Methods Fifty children with newly diagnosed T1DM and 30 healthy children（ control group） were select-ed. FOXP3 and CTLA-4 mRNA expressions were detected using real-time PCR. Soluble FOXP3 （ sFOXP3 ） and CTLA-4（sCTLA-4） protein levels in the serum were assayed by ELISA,and the levels of diabetes anti-bodies,HbA1C and C-peptide were determined using Western blotting,high performance liquid ion-exchange chromatography,and electrochemiluminescence, respectively. Results The expressions of FOXP3 mRNA and protein in T1DM patients were lower than those in the control group[0. 95 ± 0. 48 vs. 2. 11 ± 0. 79,（6. 27 ± 1. 49） ng/ml vs. （9. 02 ± 2. 37） ng/ml,respectively,both P 〈0. 01],while the expressions of CTLA-4 mRNA and protein were higher than those in the control group[2. 43 ± 0. 83 vs. 1. 94 ± 0. 84,（77. 88 ± 22. 34） ng/ml vs. （65. 97 ± 12. 11） ng/ml,respectively,both P〈0. 01]. In T1DM patients,the expressions of FOXP3 and CTLA-4 genes presented positive correlation with the protein expression（ r=0. 758,0. 396, P〈0. 05）, and FOXP3 protein expression presented correlation with CTLA-4 protein expression （ r =-0. 624,P〈0. 05）. Conclusion There are abnormal expressions of FOXP3 and CTLA-4 genes as well as their proteins in the peripheral blood of the children with newly diagnosed T1DM,and FOXP3 regulated the expression of CTLA-4 in T regulatory cells,indicating the involvement of immune mechanisms in the devel-opment of T1DM.