IFN-γ Mediates Thyroid Damage By Upregulating Fas Expression In Hashimoto Thyroiditis

Objective1.To investigate the relationship between Th1 cytokine IFN-γ and apoptotic protein Fas in thyroid tissue of Hashimoto thyroiditis.2.To investigate the regulation of Th1 cytokine IFN-γ on Fas gene expression in the rat thyroid cell lines FRTL-5.Methods1.Thyroid histological slides of HT patients were obtained from the first affiliated hospital of Anhui medical university,normal thyroid tissue adjacenting to thyroid adenoma were obtained as normal controls.The expressions and distribution of IFN-γ,Fas in Hashimoto thyrioditis and normal controls were investigated by immunohistochemical staining.2.Two intervention methods were used on cultured FRTL-5 cells in vitro.(1)FRTL-5cells were treated with IFN-γ at the final concentration of 40 ng / ml for 6,12,24,48 and 72 hours respectively for IFN-γ stimulation groups.FRTL-5 cells treated without IFN-γ were used as control group.All groups of cells were collected for later detection.(2)FRTL-5 cells were treated with IFN-γ at the final concentration of 5、10、20、40、80ng/ml respectively for IFN-γ stimulation groups.FRTL-5 cells treated without IFN-γwere used as control group.All groups of cells were collected after culturing 48 h for later detection.3.Total RNA was extracted from FRTL-5 cells using TRIzol and c DNA was synthesized using reverse transcription kits.The expression level of Fas m RNA was determined using quantitative RT-PCR.Gene expression was analyzed by relative quantification with the 2-ΔΔCtmethods.4.The expression of Fas protein in FRTL-5 cells with different dose of IFN-γ was analyzed by FACSVerse flow cytometry.Results1.The staining of IFN-γ was negative in normal thyroid tissue and the Fas-positive thyroid follicular cells was not obviously be detected.But the expressions of Fas in thyroid follicular cells and IFN-γ in infiltrating lymphocytes were more easily be detected in all HT specimens.In addition,Fas-positive thyroid follicular cells were common in lymphocyte infiltration areas,rarely expressed in areas without lymphocyte infiltration.2.The expression of Fas m RNA in FRTL-5 cells was up-regulated by IFN-γ in time-dose dependent manner.The expression of Fas m RNA in FRTL-5 cells was correlated with IFN-γ in a time-and dose-dependent manner,the difference was statistically significant at 24 h,48h and 72 h group(`x± s were 6.39± 2.02,7.45 ± 4.33,11.40± 5.05,respectively;P<0.01).The difference was statistically significant at20 ng / ml,40 ng / ml,80 ng / ml group(`x± s were 4.01 ± 1.48,10.53± 1.54,3.62± 2.22,respectively;P<0.01).3.Our data by flow cytometry showed that the positive rate of Fas protein in FRTL-5cells without the stimulation of IFN-γ was 10.1%.With the increase of IFN-γconcentration,the positive rate of Fas protein in FRTL-5 cells increased gradually,the positive rate of Fas protein in FRTL-5 cells were 28.3% in 20 ng / ml group,30.0% in40 ng / ml group and 38.9% in 80 ng / ml group,respectively.Conclusion IFN-γ secreted by infiltrating lymphocytes mediated thyroid follicular cells apoptosis by up-regulating the expressions of Fas in Hashimoto thyroiditis Thyroid tissue,thereby it may contribute to hypothyroidism in HT patients.