碘过量对实验性自身免疫性甲状腺炎大鼠甲状腺功能、抗体及TSHR基因表达的影响

The effects of iodine excess on thyroid function,antibody and TSHR gene expression in experimental autoimmune thyroiditis rats

目的建立实验性自身免疫性甲状腺炎(EAT)大鼠模型,观察碘过量对EAT大鼠甲状腺功能、抗体及促甲状腺激素受体(TSHR)基因表达的影响,探讨TSHR基因在自身免疫性甲状腺炎发生中的作用机制。方法48只4周龄雌性Lewis大鼠,按体重(80~100 g)采用随机数字表法分为对照组、甲状腺球蛋白(TG)组、TG+高碘Ⅰ(TG+HⅠ)组、TG+高碘Ⅱ(TG+HⅡ)组,每组12只大鼠,分别饮用碘离子含量为50、50μg/L和20、200 mg/L的去离子水,对TG组、TG+HⅠ组、TG+HⅡ组大鼠进行免疫,采用猪甲状腺球蛋白(pTg)和完全弗氏佐剂(CFA)作为免疫试剂,每2周1次,共3次。石蜡包埋甲状腺组织、切片,观察大鼠甲状腺病理学变化;放射免疫法检测大鼠血清甲状腺球蛋白抗体(TgAb)、甲状腺过氧化物酶抗体(TPOAb)、游离三碘甲状腺原氨酸(FT3)、游离甲状腺素(FT4)水平;酶联免疫吸附试验(ELISA)法检测大鼠血清TSHR蛋白含量;实时荧光定量PCR检测大鼠全血及甲状腺组织TSHR mRNA表达水平;免疫组织化学法检测大鼠甲状腺组织TSHR蛋白表达水平。结果HE染色显示,对照组大鼠甲状腺滤泡结构完整,形态规则,无淋巴细胞浸润;TG组可观察到少量淋巴细胞且呈现散在分布;TG+HⅠ、TG+HⅡ组滤泡结构破坏、融合,滤泡间可见小灶状淋巴细胞浸润。各组大鼠血清TgAb、TPOAb、FT3、FT4水平[中位数(四分位数间距)]比较差异有统计学意义(H=30.28、21.99、12.87、26.69,P均<0.05);与对照组比较[6.89(6.32,7.27)、11.02(7.60,12.53),5.05(2.71,7.99)、7.51(6.50,9.24)pmol/L],TG、TG+HⅠ、TG+HⅡ组大鼠血清TgAb[34.99(25.39,41.35)、37.70(29.06,43.99)、46.41(38.52,55.26)]、TPOAb[22.87(13.65,31.82)、22.22(14.82,28.33)、14.61(12.95,19.34)]、FT3[57.74(24.56,64.27)、43.64(5.69,80.03)、38.56(17.73,47.59)pmol/L]、FT4[62.16(41.22,91.57)、60.61(35.52,103.31)、47.96(31.84,112.71)pmol/L]水平明显升高(P均<0.05)。TG+HⅡ组大鼠血清TSHR蛋白表达水平[(154.26±25.95)μU/L]低于对照[(249.37±38.12)μU/L]、TG[(225.33±41.28)μU/L]、TG+HⅠ组[(218.15±65.51)μU/L,P均<0.05];与对照组比较(1.00±0.05、1.13±0.21),TG、TG+HⅠ、TG+HⅡ组大鼠全血(0.89±0.19、0.89±0.30、0.85±0.24)、甲状腺组织TSHR mRNA表达水平(0.63±0.25、0.46±0.16、0.51±0.25)明显下调(P均<0.05)。免疫组织化学染色显示,对照组大鼠甲状腺TSHR蛋白阳性强度明显高于TG、TG+HⅠ、TG+HⅡ组大鼠。结论长时间的高碘暴露可能会导致甲状腺的摄碘功能发生损伤,引发甲状腺疾病并使病情加重、TSHR基因的异常表达,使受体膜外区的促甲状腺激素结合位点具有抗原性,从而引发自身免疫性甲状腺疾病。

Objective Experimental autoimmune thyroiditis(EAT)rat model was establish to observe the effects of iodine excess on thyroid function,antibody and thyrotropin receptor(TSHR)gene expression in EAT rats,and to explore the role of TSHR gene in autoimmune thyroiditis.Methods According to body weight(80-180 g),48 rats(4-week-old female Lewis)were randomly divided into control group,thyroglobulin(TG)group,TG+high iodineⅠ(TG+HⅠ)group,and TG+high iodineⅡ(TG+HⅡ)group,12 rats per group.The iodine concentration in drinking water given to each group was 50μg/L,50μg/L,20 mg/L and 200 mg/L,respectively.At the same time,rats in TG,TG+HⅠand TG+HⅡgroups were immunized once every two weeks for three times using pTg and CFA as immunoreagent.Paraffin embedded sections of thyroid tissues were used to observe the pathological changes of rats.The serum levels of thyroglobulin antibody(TgAb),thyroperoxidase autoantibody(TPOAb),free triiodothyronine(FT3)and free thyroxine(FT4)in rats were determined by radioimmunoassay.Serum TSHR content in rats was determined by enzyme linked immunosorbent assay(ELISA).The expression of TSHR mRNA in whole blood and thyroid tissue of rats was determined by RT-PCR.The expression of TSHR protein in thyroid tissue of rats was determined by immunohistochemistry(IHC).Results Hematoxylin-eosin(HE)showed that the thyroid follicles in control group were complete in structure and regular in shape,and no lymphocyte infiltration was observed.A small number of lymphocytes were observed in TG group and scattered in distribution.Follicular structure destruction,fusion and interfollicular infiltration were observed in TG+HⅠgroup and TG+HⅡgroup.There were significant differences in serum TgAb,TPOAb,FT3 and FT4 levels among all groups(H=30.28,21.99,12.87,26.69,P<0.05).Compared to the control group[6.89(6.32,7.27),11.02(7.60,12.53),5.05(2.71,7.99),7.51(6.50,9.24)pmol/L],the levels of TgAb[34.99(25.39,41.35),37.70(29.06,43.99),46.41(38.52,55.26)],TPOAb[22.87(13.65,31.82),22.22(14.82,28.33),14.61(12.95,19.34)],FT3[57.74(24.56,64.27),43.64(5.69,80.03),38.56(17.73,47.59)pmol/L],and FT4[62.16(41.22,91.57),60.61(35.52,103.31),47.96(31.84,112.71)pmol/L]were significantly higher in TG group,TG+HⅠgroup,and TG+HⅡgroup(P<0.05).Compared with the control group[(249.37±38.12)μU/L],TG group[(225.33±41.28)μU/L],and TG+HⅠgroup[(218.15±65.51)μU/L],TSHR expression level in TG+HⅡgroup[(154.26±25.95)μU/L]were significantly decreased(P<0.05).The mRNA expression levels of TSHR gene in the whole blood(0.89±0.19,0.89±0.30,0.85±0.24)and thyroid tissue(0.63±0.25,0.46±0.16,0.51±0.25)of TG group,TG+HⅠgroup and TG+HⅡgroup were significantly lower than that of control group(1.00±0.05,1.13±0.21,P<0.05).IHC showed that the positive intensity of TSHR protein in control group was significantly higher than that in TG group,TG+HⅠgroup and TG+HⅡgroup.Conclusions Long-term exposure to high iodine will eventually lead to the damage of iodine-uptake function in thyroid gland and thyroid diseases.Abnormal expression of TSHR gene may lead to antigenicity of thyrotropin binding site in extracellular receptor region and autoimmune thyroid disease.