目的以BALB/c小鼠多次注射促甲状腺素受体(thyrotropin receptor，TSHR)膜外区A亚单位重组质粒(pcDNA3.1/TSHR289)免疫诱导Graves病(Graves′ disease，GD)伴(或)Graves眼病(Graves′ ophthalmopathy，GO)模型。
方法实验组小鼠(35只)双侧腓肠肌注射pcDNA3.1/TSHR289并电穿孔；对照组(10只)注射无菌生理盐水并电穿孔；空白组(5只)注射无菌生理盐水。各组小鼠均在第1、4、7、10周免疫，免疫前、每次免疫后2周及末次免疫后5周、8周取血检测血清TT 4、TSH、促甲状腺素受体刺激性抗体(thyrotropin receptor stimulating antibody, TSAb)、促甲状腺素受体阻断性抗体(thyrotropin receptor stimulating blocking antibody, TSBAb)。CT平扫评估小鼠眼部变化， 99mTcO 4 -显像评估甲状腺摄取功能，HE染色评估甲状腺和眼眶组织病理变化。组间均数比较采用单因素方差分析。
结果GD小鼠2次免疫后即见血清TT 4、TSAb和TSBAb均持续显著高于对照组和空白组(分别 F＝13.781，31.435，36.112，均 P<0.01)，TSH持续低于对照组和空白组( F＝13.966， P<0.01)。4次免疫后GD小鼠甲状腺 99mTcO 4 -摄取能力明显增强，甲状腺肿大，腺内大量淋巴细胞浸润，滤泡胶质稀薄。GO小鼠眼CT扫描见眼外肌增粗肿胀，肌腱及肌附着点未见异常。眼外肌和球后组织可见淋巴细胞、透明质酸和脂肪细胞浸润等病理改变。
结论BALB/c小鼠多次肌肉注射pcDNA3.1/TSHR289可成功诱导GD伴(或)GO动物模型。
ObjectiveTo construct a model of Graves′ disease(GD) with (or) Graves′ ophthalmopathy(GO) in BALB/c mice by immunization with pcDNA3.1/TSHR289.
MethodspcDNA3.1/TSHR289 was injected into the bilateral gastrocnemius muscle of 35 model mice and electroporation was immediately performed. 10 control mice were injected with sterile saline and electroporated, while 5 blank mice were injected with sterile saline only. Each group of mice was immunized at 1, 4, 7, and 10 weeks, respectively. Serum total T 4, TSH, TSAb, and TSBAb were measured before immunization, 2 weeks after each immunization, as well as 5 and 8 weeks after the last immunization. CT scan was used to evaluate the morphological changes of the eyes of the mice. 99mTcO 4 - imaging was used to measure the thyroid uptake function, and the pathological changes of the thyroid and orbital tissues were evaluated by HE staining.
ResultsAfter the 2nd time immunization, the serum concentrations of TT 4, TSAb and TSBAb in GD mice were significantly higher than those of control and blank groups( F=13.781, 31.435, 36.112, P<0.01, respectively). The TSH continued to be significantly lower than that of control and blank groups( F=13.966, P<0.01). After the 4th time immunizations, the ability of uptaking 99mTcO 4 - in GD mice thyroid was significantly enhanced compared with the control group. The thyroid goiter with a large amount of lymphocyte infiltration, and the thyroid follicle was thin. CT scan of GO mice showed thickening and swelling of the extraocular muscles, and no abnormalities in tendon and muscle attachment points. HE staining showed thickening of extraocular muscle fibers, lymphocyte infiltration of extraocular muscles and orbital tissue, increased hyaluronic acid, and infiltration of fat cells.
ConclusionGD or GO model can be successfully induced by multiple intramuscular injection of pcDNA3.1/TSHR289 in BALB/c mice.
Li Ning and Feng Siyuan are contributed equally to the article.
李宁,冯思源,孙磊,等. TSHR基因免疫诱导BALB/c小鼠Graves甲亢伴Graves眼病的实验研究[J]. 中华内分泌代谢杂志,2019,35(5)：417-424.
DOI:10.3760/cma.j.issn.1000-6699.2019.05.012版权归中华医学会所有。
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李宁和冯思源对本文有同等贡献
李宁，重组质粒构建、动物模型制备、血清学指标检测、组织取材、论文撰写；冯思源，动物模型制备、数据整理、动物显像、论文撰写；孙磊，小动物SPECT/CT、CT显像；郑薇，研究指导、论文修改、经费支持；王深，小动物SPECT/CT显像；申一鸣，小动物SPECT/CT显像；林晓云，组织病理学实验；王萱，动物饲养、取血、数据整理；孟召伟，统计学分析；贾强，小动物显像技术指导；谭建，研究指导、论文修改；宋其韬，研究指导
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