背景 甲状腺相关眼病(TAO)是眼科临床常见的难治性疾病,发病率在眼眶疾病中居首位.目前对于TAO的研究缺乏可复制的动物模型,在疾病早期阶段难以获得眼眶组织,具体病因及确切的发病机制仍未得到阐明.要详细了解TAO的发病机制,探寻有效防治措施,关键在于建立适当的动物模型. 目的采用脂质体包裹的促甲状腺激素受体(TSHR)胞外段基因重组质粒免疫同系雌性BALB/c小鼠,探讨该方法建立TAO动物模型的可行性. 方法 按照计算机随机分组方法将32只同系6～8周龄雌性BALB/c小鼠随机分为空白对照组、空质粒注射组、脂质体注射组和重组质粒注射组.分别于0、3、6周免疫各组小鼠,重组质粒注射组小鼠经双胫前肌和腹腔内分别注射阳离子脂质体包裹的pcDNA3.1 +/hTSHR289 30 μg和40 μg;脂质体注射组小鼠分别经双胫前肌和腹腔内分别注射未包裹重组质粒的阳离子脂质体30 μg和40 μg;空质粒注射组小鼠分别经双胫前肌和腹腔内分别注射pcDNA3.1+空质粒30 μg和40 μg;空白对照组小鼠未行任何干预.分别于初次免疫前及初次免疫后1、2、3、4个月测量各组小鼠的体质量,观察小鼠外眼表现.于初次免疫后17周末处死小鼠,取甲状腺及眼眶组织进行组织病理学观察;收集各组小鼠心脏血0.6 ～0.8 ml,采用ELISA法测定小鼠血清总甲状腺素4(TT4)和促甲状腺激素(TSH)的质量浓度. 结果 重组质粒注射组6只小鼠12只眼出现眼球突出、眼睑肿胀和角膜溃疡.空白对照组、脂质体注射组、空质粒注射组小鼠随着时间的延长体质量逐渐增加,而重组质粒注射组小鼠体质量则逐渐下降,总体比较差异有统计学意义(F时间=3.838,P=0.023),不同组间小鼠体质量总体比较差异有统计学意义(F分组=3.425,P=0.028),其中注射后2、3、4个月重组质粒注射组小鼠体质量明显低于空白对照组、空质粒注射组和脂质体注射组,差异均有统计学意义(均P＜0.05).空白对照组、脂质体注射组、空质粒注射组和重组质粒注射组小鼠血清TT4质量浓度分别为(7.75±1.00)、(7.96±0.76)、(6.76±1.10)和(4.43±2.88) μg/dl,TSH质量浓度分别为(6.36±2.58)、(4.83±3.96)、(6.63±1.71)和(1.60±1.76) ng/ml,总体比较差异均有统计学意义(F=7.150,P＜0.001;F=5.521,P＜0.01),其中重组质粒注射组小鼠血清中TT4和TSH质量浓度均明显低于空白对照组、脂质体注射组、空质粒注射组,差异均有统计学意义(均P＜0.05).组织病理学检查显示,6只重组质粒注射组小鼠甲状腺出现淋巴细胞浸润,15只眼小鼠眼眶组织出现眼眶内脂肪组织增生、淋巴细胞及肥大细胞浸润、透明质酸沉积以及眼外肌肌纤维肿胀、变性和断裂,并伴有炎性细胞浸润.结论 采用脂质体包裹的TSHR胞外段基因重组质粒免疫同系雌性BALB/c小鼠建立TAO动物模型是一种可行、有效的方法,该模型与人TAO的病理组织学特征相似,成模率高.%Background Thyroid-associated ophthalmopathy (TAO) is a kind of clinically common and incurable ocular disease,and its incidence is at top place.The etiology and pathologic mechanism of TAO are still unknown because of shortness of replicative animal models and difficulty to acquire the ocular tissues in the early stage of the disease.To better understand the pathogenesis of TAO and investigate effective treatable measures, an appropriate animal model should be developed.Objective This study was to immunize female BALB/c mice with the recombinant plasmid of human thyroid-stimulating hormone receptor (TSHR) extracellular domain in cationic liposomes for the establishement of TAO models.Methods Thirty-two 6-to 8-week-old female BALB/c mice were randomly assigned to four groups according to computer random allocation.pcDNA3.1 +/hTSHR289 of 100 μg in an adjuvant cationic liposomes was injected via anterior tibialis muscle and peritoneal cavity separately in the recombinant plasmid injection group in 0, 3,6 weeks, and pcDNA3.1 or cationic liposomes was injected in the liposomes injection group or the blank plasmid group in the same way, respectively, and normal saline solution was injected in the blank control group.Body weight of the mice was measued before and 1 month,2,3 and 4 months after initial injection.The manifestations were observed after modeling.The mice were sacrificed 17 weeks after initial injection,and the histopathology examination was carried out on the thyroid gland and orbital tissue.The heart blood was collected from the mice,and serum contents of total thyroxin 4 (TT4) and thyroid-stimulating hormone (TSH)were assayed by ELISA.Results Protrusion, eyelid swell and keratitis occurred in 12 eyes of 6 mice in the recombinant plasmid injection group after immunization.A significant difference in the body weight of the mice was found among the blank control group, blank plasmid group, liposomes injection group and recombinant plasmid injection group (Fgroup =3.425, P =0.028), and the body weight was considerably reduced in the recombinant plasmid injection group in comparison with the blank control group, blank plasmid group,liposomes injection group (Ftime =0.838 ,P=0.023).The serum levels of TT4 were (7.75±1.00), (7.96±0.76), (6.76±1.10) and (4.43±2.88) μg/dlin the blank control group, liposomes injection group, blank plasmid group, and recombinant plasmid injection group, and those of TSH were (6.36±2.58),(4.83±3.96),(6.63±1.71) and (1.60 ±1.76) ng/ml, showing significant differences among the groups (F =7.150, P＜0.001;F =5.521, P＜0.01) , and the serum levels of TT4 and TSH were remarkably lower in the recombinant plasmid injection group than those of the blank control group,liposomes injection group and blank plasmid group (all at P ＜ 0.05).Histopathology revealed the lymphocyte infiltration of thyroid gland in 6 mice and proliferation of orbital adipose tissue, infiltration of lymphocytes and mastocytes,deposition of hyaluronic acid as well as swell, breakage and inflammatory cell infiltration of extraocular muscle in 15 eyes of the recombinant plasmid injection group.Conclusions A murine model of TAO can be successfully induced by immunization with recombination plasmid pcDNA3.1 +/hTSHR289 and cationic liposomes.The histopathology characteristics and ocular findings of the animal models are similar to human TAO.
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