Abstract：
OBJECTIVE: The study aimed to establish a mouse model of Graves\' disease (GD) with Graves\' orbitopathy (GO; GD + GO) that can represent the clinical disease characteristics.
METHODS: A eukaryotic expression plasmid of insulin-like growth factor 1 receptor (IGF-1R) α subunit (pcDNA3.1/IGF-1Rα) and a thyrotropin receptor (TSHR) A subunit plasmid (pcDNA3.1/TSHR-289) were injected in female BALB/c mice followed by immediate electroporation to induce a GD + GO model. Grouping was performed according to the frequency of injection (2- to 4-week intervals) and type of injected plasmids: T: pcDNA3.1/TSHR-289( +), I: pcDNA3.1/IGF-1Rα( +), or co-injection T + I: pcDNA3.1/TSHR-289( +) and pcDNA3.1/IGF-1Rα( +). Serum TSH, T4, TSAb, TSBAb, body weight, and blood glucose levels were evaluated. Thyroid 99mTcO4- imaging and retrobulbar magnetic resonance imaging (MRI) were performed, and bilateral eye muscle volumes were measured. Immunohistochemistry and hematoxylin-eosin staining were performed on the relevant tissues, and semi-quantitative analysis was performed.
RESULTS: A total of 60% of mice (3/5, one mouse died) in the T group developed GD + GO. In the T + I group, 83.3% of mice (5/6) developed GD + GO. Mice in the I group did not develop GD. Compared with the control group, serum T4, TSAb, and TSBAb of the mice in the GD + GO model groups were increased to varying degrees (P < 0.05), and serum TSH and body weight were significantly lower compared to the control group (P < 0.05). The thyroid uptake capacity of 99mTcO4- and the volume of eye muscle of mice in the GD + GO group were significantly higher compared to the control group (P < 0.05). The thyroid and retrobulbar muscles of these mice showed varying inflammatory infiltration and interstitial muscle edema. The severity of GD + GO in the co-injection group was not related to injection frequency; however, GD and ocular signs in co-injection mice were more severe compared to the T group.
CONCLUSIONS: We successfully induced a GD + GO mouse model by a repeated co-injection of pcDNA3.1/IGF-1Rα and pcDNA3.1/TSHR-289 plasmids. Injection of pcDNA3.1/IGF-1Rα alone failed to induce GD. Co-injection of two plasmids induced more severe GD + GO than pcDNA3.1/TSHR-289( +) alone.
METHODS: A eukaryotic expression plasmid of insulin-like growth factor 1 receptor (IGF-1R) α subunit (pcDNA3.1/IGF-1Rα) and a thyrotropin receptor (TSHR) A subunit plasmid (pcDNA3.1/TSHR-289) were injected in female BALB/c mice followed by immediate electroporation to induce a GD + GO model. Grouping was performed according to the frequency of injection (2- to 4-week intervals) and type of injected plasmids: T: pcDNA3.1/TSHR-289( +), I: pcDNA3.1/IGF-1Rα( +), or co-injection T + I: pcDNA3.1/TSHR-289( +) and pcDNA3.1/IGF-1Rα( +). Serum TSH, T4, TSAb, TSBAb, body weight, and blood glucose levels were evaluated. Thyroid 99mTcO4- imaging and retrobulbar magnetic resonance imaging (MRI) were performed, and bilateral eye muscle volumes were measured. Immunohistochemistry and hematoxylin-eosin staining were performed on the relevant tissues, and semi-quantitative analysis was performed.
RESULTS: A total of 60% of mice (3/5, one mouse died) in the T group developed GD + GO. In the T + I group, 83.3% of mice (5/6) developed GD + GO. Mice in the I group did not develop GD. Compared with the control group, serum T4, TSAb, and TSBAb of the mice in the GD + GO model groups were increased to varying degrees (P < 0.05), and serum TSH and body weight were significantly lower compared to the control group (P < 0.05). The thyroid uptake capacity of 99mTcO4- and the volume of eye muscle of mice in the GD + GO group were significantly higher compared to the control group (P < 0.05). The thyroid and retrobulbar muscles of these mice showed varying inflammatory infiltration and interstitial muscle edema. The severity of GD + GO in the co-injection group was not related to injection frequency; however, GD and ocular signs in co-injection mice were more severe compared to the T group.
CONCLUSIONS: We successfully induced a GD + GO mouse model by a repeated co-injection of pcDNA3.1/IGF-1Rα and pcDNA3.1/TSHR-289 plasmids. Injection of pcDNA3.1/IGF-1Rα alone failed to induce GD. Co-injection of two plasmids induced more severe GD + GO than pcDNA3.1/TSHR-289( +) alone.
摘要：
目的：本研究旨在建立能够代表临床疾病特征的Graves病（GD）伴Graves眼眶病（GO；GDGO）小鼠模型。
方法：将胰岛素样生长因子1受体（IGF-1R）α亚基（pcDNA3.1/IGF-1Rα）和促甲状腺激素受体（TSHR）A亚基质粒（pcDNA3.1/TSHR-289）的真核表达质粒注射到雌性BALB/c小鼠中，然后立即电穿孔以诱导GDGO模型。根据注射频率（2至4周间隔）和注射质粒类型进行分组：T：pcDNA3.1/TSHR-289（），I:pcDNA3.1/IGF-1Rα(+),或共注射T+I:pcDNA3.1/TSHR-289(+)和pcDNA3.1/IGF-1Rα(+)。血清TSH,T4、TSAb、TSBAb,体重,并评估血糖水平。进行甲状腺99mTcO4-成像和球后磁共振成像（MRI），测量双侧眼肌体积。对相关组织进行免疫组织化学和苏木精-伊红染色，并进行半定量分析。
结果:T组中总共有60%的小鼠(3/5,一只小鼠死亡)出现GD+GO。在T+I组中,83.3%的小鼠(5/6)发展为GD+GO。I组中的小鼠没有发展GD。与对照组相比,血清T4，TSAb，GD+GO模型组小鼠的TSBAb均有不同程度的升高(P<0.05),血清TSH和体重均显著低于对照组(P<0.05)。GD+GO组小鼠99mTcO4-的甲状腺摄取能力和眼肌体积明显高于对照组(P<0.05)。这些小鼠的甲状腺和球后肌肉显示出不同的炎症浸润和间质肌肉水肿。共注射组GD+GO的严重程度与注射频率无关；然而，与T组相比,共注射小鼠中的GD和眼部体征更严重。
结论：我们通过重复共注射pcDNA3.1/IGF-1Rα和pcDNA3.1/TSHR-289质粒成功诱导了GD+GO小鼠模型。单独注射pcDNA3.1/IGF-1Rα不能诱导GD。两个质粒的共注射比单独的pcDNA3.1/TSHR-289(+)诱导更严重的GD+GO。
方法：将胰岛素样生长因子1受体（IGF-1R）α亚基（pcDNA3.1/IGF-1Rα）和促甲状腺激素受体（TSHR）A亚基质粒（pcDNA3.1/TSHR-289）的真核表达质粒注射到雌性BALB/c小鼠中，然后立即电穿孔以诱导GDGO模型。根据注射频率（2至4周间隔）和注射质粒类型进行分组：T：pcDNA3.1/TSHR-289（），I:pcDNA3.1/IGF-1Rα(+),或共注射T+I:pcDNA3.1/TSHR-289(+)和pcDNA3.1/IGF-1Rα(+)。血清TSH,T4、TSAb、TSBAb,体重,并评估血糖水平。进行甲状腺99mTcO4-成像和球后磁共振成像（MRI），测量双侧眼肌体积。对相关组织进行免疫组织化学和苏木精-伊红染色，并进行半定量分析。
结果:T组中总共有60%的小鼠(3/5,一只小鼠死亡)出现GD+GO。在T+I组中,83.3%的小鼠(5/6)发展为GD+GO。I组中的小鼠没有发展GD。与对照组相比,血清T4，TSAb，GD+GO模型组小鼠的TSBAb均有不同程度的升高(P<0.05),血清TSH和体重均显著低于对照组(P<0.05)。GD+GO组小鼠99mTcO4-的甲状腺摄取能力和眼肌体积明显高于对照组(P<0.05)。这些小鼠的甲状腺和球后肌肉显示出不同的炎症浸润和间质肌肉水肿。共注射组GD+GO的严重程度与注射频率无关；然而，与T组相比,共注射小鼠中的GD和眼部体征更严重。
结论：我们通过重复共注射pcDNA3.1/IGF-1Rα和pcDNA3.1/TSHR-289质粒成功诱导了GD+GO小鼠模型。单独注射pcDNA3.1/IGF-1Rα不能诱导GD。两个质粒的共注射比单独的pcDNA3.1/TSHR-289(+)诱导更严重的GD+GO。
