Study on the immunological mechanism of anti-TSH receptor antibody production by using transgenic mice

在Graves病中，针对TSH受体的自身抗体（TSHR）模拟TSH的作用，因此称为甲状腺刺激抗体（TSAb）。TSAb引起甲状腺过度刺激和甲状腺功能亢进。动物模型是阐明自身免疫性甲状腺疾病病理生理机制的重要工具，近年来已建立了几种Graves病动物模型，其中一些采用了基因免疫。建立了在人角质形成细胞基因启动子控制下表达Cre重组酶的转基因小鼠系。通过使用北方印迹检测角蛋白驱动的Cre重组酶的活性和特异性。该转基因小鼠在角质形成细胞中特异性地缺失loxP插入的lac Z基因，并在发育期间和/或在成年角质形成细胞中诱导TSHR基因。然而，我们没有检测到抗TSHR抗体的活性和高水平的血清甲状腺激素，因此，我们使用电穿孔（EP）技术建立了一种新的Graves病模型，期望在体内大大提高hTSHR的表达。在非病毒的质粒载体体内基因转移技术中，直接肌肉注射质粒DNA的方法操作简单，但受转染基因表达水平相对较低的限制，我们建立了hTSHR稳定表达克隆（AHK 12），对bTSH有功能反应，用于检测自身抗体的生物学活性和ELISA用于检测针对rhTSHR-289 His（对应于细胞外A亚基TSHR）的自身抗体。

In Graves' disease, autoantibody directed against TSH receptor (TSHR) mimic the action of TSH and are therefore called thyroid-stimulating antibody (TSAb). TSAb caused overstimulation of the thyroid gland and hyperthyroidism. Animal models are very useful tools to clarify the pathophysiology of autoimmune thyroid disease.Recently, several animal models of Graves' disease have been developed, some of which employed genetic immunization. A transgenic mouse line that expresses Cre recombinase under control of the human keratinocyte gene promoter was established. The activity and specificity of the keratin-driven Cre recombinase were examined by using Northern blotting. This keratin-Cre transgenic mouse was specifically delete loxP-inserted lac Z gene in keratinocytes and induced TSHR gene during development and/or in adult keratinocytes. However, we could not detect the activities of anti-TSHR antibody and high levels of serum thyroid hormone.We then used the technique of electroporation (EP) for establishment of a new model of Graves' disease in expectation of greatly enhanced hTSHR expression in vivo. Among non-viral techniques for gene transfer by plasmid vector in vivo, the method with direct injection of plasmid DNA into muscles is simple, while is restricted by the relatively low expression levels of the transferred gene.Moreover, we established hTSHR expressing stable clone (AHK12), having functional response to bTSH, for the detection of biological activity of autoantibody and ELISA for the detection of autoantibody against rhTSHR-289His, corresponding to the extracellular A subunit TSHR.