| Part I The expressions of Treg and Th17 cells associated cytokines in Graves9 disease patients before and after 131I treatmentObjective:This study aimed to detect the expressions of Treg and Th17 cells associated cytokines in peripheral blood mononuclear cells in Graves'disease patients before and after 131I treatment, and explore the pathological mechanism of these cells in the development of GD.Methods:The mRNA levels of Foxp3, IL-17, IL-23, IL-27 and IFN-y in PBMCs were measured by quantitative real-time PCR (QRT-PCR) from 40 healthy controls and 45 GD patients before and after 131I treatment. Thyroid function and autoantibody levels in GD were performed by electrochemiluminescence. The correlation analysis was performed by SPSS 17.0.Results:The Foxp3 gene expression level in PBMC was significantly lower in GD patients before 131I treatment compared with healthy controls (P<0.0001). In addition, the levels of IL-17, IL-23, IL-27 and IFN-y were higher in GD than healthy controls (P<0.0001). After 131I treatment, Foxp3 level was increased slightly (P=0.0391), but still lower in GD compared with healthy controls (P=0.0150). On the contrary, the levels of IL-17, IL-23 and IL-27 were decreased (P<0.05), but still higher than healthy controls (P<0.001). The level of IFN-y showed a recovery. The correlation analysis showed that the level of Foxp3 was negatively correlated with serum levels of FT3, FT4 and TRAb. Besides, the level of Foxp3 was negatively correlated with IL-17 expression level. Additionally, IL-17 and IL-23 expression levels were positively correlated with FT3 and FT4. IL-23 and IL-27 were positively correlated with TRAb.Conclusion:The cytokines of Treg and Th17 cells may play an important role in the development of GD.131I treatment could work on GD via these cytokines.Part II Transcription factors of Tregs-Foxp3 gene polymorphisms associated with susceptibility of Graves'disease in Chinese Han populationObjective:To investigate the association between Foxp3 gene polymorphisms and the susceptibility to GD, four single nucleotide polymorphisms (SNP) including -2383,-3279,-3499 in the promoter and ?S9+459 in the intron were genotyped.Methods:Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 308 GD patients and 306 healthy controls. The relative expression level of Foxp3 gene was measured by qRT-PCR.Results:At the -3279 site, A carriers (AA+CA) were significantly higher in the cases than the controls (36.4%vs 26.5%, P=0.008). The frequency of Foxp3-3279 A allele was significantly higher in GD (21.4% vs 15.0%, P=0.004). At the same time, the frequencies of AA/CA genotypes in female GD were higher than male GD. For the Foxp3 ?S9+459 polymorphism, the CC genotype was higher in GD than the controls (P=0.006) and the CC genotype was associated with a higher risk of GD (adjusted OR,2.781; 95%CI, 1.332-6.187). There was no significant difference between GD patients and controls for-2383C/T and-3499A/G (P=0.597 and P=0.784). In the haplotypes analysis, we found that the frequencies of CCA, CAA and TCA haplotypes were significantly different between GD patients and healthy controls (P<0.0001, P=0.0008 and P<0.0001). The CAA and TCA haplotypes, with higher frequencies in GD, showed an increased risk of GD (16.5% vs 10.2%, OR=1.777,95% CI 1.266-2.494 and 6.5% vs 1.7%, OR=3.979,95% CI 2.001-7.913). In the meantime, the haplotype CCA was associated with a decreased susceptibility to GD (54.1% vs 65.4%, OR=0.610,95% CI 0.478-0.778).Conclusion:-3279 and ?S9+459 polymorphisms were associated with the susceptibility to GD in Chinese Han population, but-2383 and -3499 sites were not. The CAA and TCA haplotypes could be as risk factors to increase the susceptibility to GD. On the contrary, the haplotype CCA appeared a protective factor to decrease the risk of GD.Part? The functional changes caused by Foxp3 gene polymorphisms in GD patientsObjective:Because of the association of Foxp3 gene polymorphisms and GD, this study aimed to investigate the functional changes result from Foxp3 gene polymorphisms in GD patients.Methods:Foxp3 DNA fragments from GD patients with different haplotypes were cloned in pGL3-Basic vector, we acquired pGL3-CCA (allele C) and pGL3-CAA (allele A) plasmid constructs. HeLa and 293T cells were cotransfected with pGL3-CCA or pGL3-CAA to detective the luciferase activity of Foxp3 promoter. Foxp3 expression level in GD with different genotypes was measured by RT-PCR. In the meantime, thyroid function and autoantibody levels in GD with different genotypes were performed by electrochemiluminescence.Results:The expression of Foxp3 in GD with AA/CA genotype of-3279 was lower than GD with CC genotype (P=0.004). The FT3 level of GD patients with-3279 CA genotype was lower than GD patients with AA/CC genotype (P=0.045, P=0.018). GD patients with higher TSH level and/or lower TRAb level were more frequent to carry-3279 A allele (P=0.014, P=0.017). For the Foxp3-2383, the higher FT3 level occurred more frequently in GD patients with TT genotype (P=0.032, P=0.021). In addition, GD patients with higher TGAb level were more frequent to carry-3499A allele (P=0.029). The relative luciferase activity of pGL3-CAA was lower than that of pGL3-CCA in 293T cells (P=0.030) and HeLa cells (P=0.011).Conclusion:For the mutation from C to A at position of-3279, the reporter activity was reduced by more than 40%. At the same time, the relative Foxp3 gene expression level was decreased. In addition, the thyroid function and autoantibody levels in GD patients were changed too. |
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