采用人促甲状腺激素受体活化的脾细胞免疫小鼠建立甲状腺相关眼病动物模型的研究

目的采用人促甲状腺激素受体活化的脾细胞免疫同源BALB/c小鼠建立甲状腺相关眼病(TAO)动物模型。方法将23只BALB/c小鼠预处理后用完全随机设计的方法分为2组:重组质粒免疫组(A组)18只,空质粒免疫组(B组)5只,分别接受重组质粒pcDNA3·1/hTSHR和空质粒pcDNA3·1免疫,免疫后第3、6周加强免疫2次。18周后处死所有小鼠,机械分离脾细胞,取眼眶组织做病理学观察。另取26只BALB/c小鼠用完全随机设计的方法分为2组:活化脾细胞免疫组(C组)16只和未活化脾细胞免疫组(D组)10只,分别接受A组和B组的脾细胞免疫。4周后处死所有小鼠,取眼眶组织作病理学观察,用放射免疫法测定血清总甲状腺素水平,免疫放射法测定血清促甲状腺激素水平,酶联免疫吸附法测定血清促甲状腺激素受体抗体。结果A组:约25%小鼠眼眶组织出现黏液变性、水肿,无明显纤维组织和脂肪组织增生,肌纤维无变性断裂。C组:约50%小鼠眼眶组织出现明显黏液变性、水肿,局部纤维组织和脂肪组织增生,肌纤维透明变性断裂;电镜下可见滑面内质网扩张,肌原纤维排列紊乱。B组和D组:小鼠的眼眶组织均无形态学改变。脾细胞免疫后4周,血清总甲状腺素:C组(7·130±1·017)μg/dl与D组(6·431±0·573)μg/dl相比显著升高,差异有统计学意义(P=0·036);血清促甲状腺激素:C组(0·070±0·032)μIU/ml与D组(0·098±0·020)μIU/ml相比显著降低,差异有统计学意义(P=0·020);血清促甲状腺激素受体抗体:C组(0·202±0·067)与D组(0·151±0·055)相比无显著升高,差异无统计学意义(P=0·055)。结论采用人促甲状腺激素受体活化的脾细胞免疫同源BALB/c小鼠所构建的动物模型与人TAO的组织学特征非常接近,是一种可行、有效的方法。促甲状腺激素受体和针对该受体的T淋巴细胞在其自身免疫反应过程中具有重要作用。

Establishment of an animal model for thyroid associated ophthalmopathy by treatment of mice with human thyrotropin receptors-activated splenocytes

OBJECTIVE: To establish an animal model for thyroid-associated ophthalmopathy (TAO) by treatment of homoimmune BALB/c mice with human thyrotropin receptors ( hTSHR )-activated splenocytes. METHODS Twenty three BALB/c mice were randomly divided into group A and B, which were immune with recombinant plasmid pcDNA3. 1/hTSHR or blank plasmid pcDNA3. 1 for 3 times at 3-week intervals, respectively. Mice in these two groups were sacrificed 18 weeks after immunization and the splenocytes were isolated. Other 26 homoimmune BALB/c mice were divided randomly into group C and group D which received splenocytes from group A and group B, respectively. Four weeks later the orbital tissues were harvested for pathological examination and serum was collected for TT4, TSH and TRAb measurements. RESULTS After immunization with recombinant plasmid pcDNA3. l/hTSHR, orbital tissues displayed edema and mucinous degeneration in 25% of mice but no proliferation of adipose tissue and fibrous tissue (group A). After immunization with hTSHR-activated splenocytes, orbital tissues displayed proliferation of adipose tissues and fibrous tissues, degeneration and disruption of muscular fibers microscopically in 50% of mice (group C). Under electronic microscope, expansion of smooth endoplasmic reticulum and disorder of myofibrils were found in these mice. In all mice from group B and group D, orbital tissues were normal histologically. After immunization with splenocytes, serum total T4 levels were significantly elevated ( P = 0. 036) in group C (7. 130+/-1.017) microg /dl when compared with group D (6. 431+/-0. 573) microg /dl. Serum TSH levels were significantly reduced (P = 0. 020) in group C (0. 070+/-0. 032) microIU/ml when compared with group D (0. 098+/-0. 020) microIU/ml. Serum TRAb levels were slightly increased in group C(0. 202 +/-0. 067) as compared to group D(0. 151+/-0. 055) , however, this difference was statistically non-significant (P = 0. 055 ). CONCLUSIONS: TAO animal model established by immunizing homoimmune BALB/c mice with hTSHR-activated splenocytes is similar to human TAO in histological characteristics and is a feasible and reliable experimental method. Our study also proves that the thyrotropin receptor and T cell aiming directly at this auto-antigen play important roles in the autoimmune process of TAO.