An improved Graves' disease model established by using in vivo electroporation exhibited long-term immunity to hyperthyroidism in BALB/c mice
An improved Graves' disease model established by using in vivo electroporation exhibited long-term immunity to hyperthyroidism in BALB/c mice
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DOI：
10.1210/en.2006-1077
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发表时间：
2007-05-01
期刊：
影响因子：
4.8
通讯作者：
Yoshida, Tadashi
中科院分区：
文献类型：
--
作者：
Kaneda, Toshio;Honda, Asako;Yoshida, Tadashi
在Graves病中，甲状腺的过度刺激和甲状腺功能亢进是由针对TSH受体（TSHR）的自身抗体引起的，TSHR模仿TSH的作用。动物模型的建立是研究自身免疫性甲状腺机能亢进病理生理和免疫学分析的重要步骤。在本研究中，我们采用电穿孔（EP）技术进行基因免疫，实现了人TSHR （hTSHR）在体内表达的显著增强和对小鼠甲亢的有效诱导。在一项使用β -半乳糖苷酶（β -gal）表达载体的初步研究中，通过EP导入肌肉的β -gal酶活性比之前通过直接基因转移方法导入的高40倍以上。通过RT-PCR，可以检测到EP转移的cDNA在肌肉组织中持续表达hTSHR mRNA至少2周。基于这些结果，我们通过插入hTSHR或hTSHR289His cDNA的两种表达载体诱导甲状腺功能亢进。结果表明，经3 ~ 4次EP重复免疫后，hTSHR免疫的BALB/c小鼠（12.0 ~ 31.8%）和hTSHR289His免疫的BALB/c小鼠（79.2 ~ 95.7%）由于tshr刺激抗体的作用，总T-4水平升高，甲状腺滤泡增生，上皮高度不规则。此外，令人惊讶的是，tshr刺激抗体在最后一次免疫后持续了8个多月。这些结果表明，体内EP基因免疫比以前的方法更有效，并且有助于描述Graves病的病理生理。
In Graves' disease, the overstimulation of the thyroid gland and hyperthyroidism are caused by autoantibodies directed against the TSH receptor (TSHR) that mimics the action of TSH. The establishment of an animal model is an important step to study the pathophysiology of autoimmune hyperthyroidism and for immunological analysis. In this study, we adopted the technique of electroporation (EP) for genetic immunization to achieve considerable enhancement of in vivo human TSHR (hTSHR) expression and efficient induction of hyperthyroidism in mice. In a preliminary study using beta-galactosidase (beta-gal) expression vectors, beta-gal introduced into the muscle by EP showed over 40-fold higher enzymatic activity than that introduced via previous direct gene transfer methods. The sustained hTSHR mRNA expression derived from cDNA transferred by EP was detectable in muscle tissue for at least 2 wk by RT-PCR. Based on these results, we induced hyperthyroidism via two expression vectors inserted with hTSHR or hTSHR289His cDNA. Consequently, 12.0 - 31.8% BALB/c mice immunized with hTSHR and 79.2-95.7% immunized with hTSHR289His showed high total T-4 levels due to the TSHR-stimulating antibody after three to four times repeated immunization by EP, and thyroid follicles of which were hyperplastic and had highly irregular epithelium. Moreover, TSHR-stimulating antibody surprisingly persisted more than 8 months after the last immunization. These results demonstrate that genetic immunization by in vivo EP is more efficient than previous procedures, and that it is useful for delineating the pathophysiology of Graves' disease.