摘要目的 通过免疫电穿孔的方法,构建稳定的BALB/c小鼠(简称小鼠)格雷夫斯病(GD)模型.方法 制备重组质粒pcDNA3.1/TSHR268.将50只小鼠按随机数字表法分为实验组(30只)、对照组(10只)和空白组(10只).分别于实验第1、4、7、10周在实验组小鼠双后肢腓肠肌注射重组质粒,在对照组及空白组小鼠相同位置注射相同体积生理盐水;实验组及对照组小鼠每次注射后在注射区域行电穿孔加强免疫.以放射免疫法检测小鼠血清T4,ELISA法检测小鼠TRAb氨基端(TRAb N)抗体和TRAb羧基端(TRAb C)抗体.对模型小鼠进行甲状腺99TcmO4显像及甲状腺形态学、病理学分析.多组间均数比较采用单因素方差分析及最小显著差异t检验.结果 实验组建模成功率为80％(24/30).24只成功建模的BALB/c小鼠血清T4由第0周的(16.06±5.16) nmol/L升高至第12周的(95.04±68.92) nmol/L(F=18.906,t=-5.598,P＜0.05),TRAb N抗体由第0周的(0.006±0.002) U/L升高至第12周的(0.251±0.110) U/L(F=47.491,t =-10.869,P＜0.05),TRAbC抗体由第0周的(11.176±2.635) ×103任意单位(AU)/L升高至第12周的(46.395±22.001)×103 AU/L(F=14.642,t=-7.787,P＜0.05).停止免疫后的第18周,小鼠血清T4为(36.64±23.68) nmoL/L、TRAb N抗体为(0.094±0.053) U/L、TRAb C抗体为(24.456±6.725)×103 AU/L,均有所下降,但仍明显高于免疫前水平(t=-4.161、-8.085和-9.008,均P＜0.05).对照组与空白组小鼠T4、TRAb N抗体及TRAb C抗体在免疫前后均未见明显变化.经过4次免疫,实验组小鼠甲状腺对99TcmO4-的摄取能力较对照组及空白组明显增强.GD模型小鼠甲状腺较正常BALB/c小鼠体积增大,病理学检查可见甲状腺组织淋巴细胞浸润.结论 重组质粒pcDNA3.1/TSHR268+免疫电穿孔方法可成功构建BALB/c小鼠GD模型.

abstractsObjective To establish stable Graves disease (GD) mice models with immunization and electroporation (EP).Methods Fifty mice were divided into 3 groups by random number table method:experimental group (n =30),control group (n =10),blank group (n =10).Recombinant plasmid pcDNA3.1/hTSHR268 was constructed and injected to bilateral gastrocnemius in experimental group mice on the 1st,4th,7th and 10th week.The same volume of normal saline was injected in the control group and blank group at the same time.Both experimental group and control group were subjected to EP at the same time and the same location to enhance immunization.Serum T4 was tested with radioimmunoassay.TRAb N-terminal (TRAb N) and TRAb C-terminal (TRAb C) antibodies were tested with ELISA.Whole body 99TcmO4-imaging was performed and then thyroid morphology and pathology were investigated.Data were analyzed by one-way analysis of variance and the least significant difference (LSD) t test.Results GD BALB/c mice models were built successfully (80％,24/30).Serum T4 increased from (16.06±5.16) nmol/L at the basic level to(95.04±68.92) nmol/L on the 12th week(F=18.906,t=-5.598,P＜0.05).Serum TRAb N antibody increased from (0.006±0.002) U/L at the basic level to (0.251±0.110) U/L on the 12th week(F=47.491,t=-10.869,P＜0.05).Serum TRAb C antibody increased from (11.176±2.635)×103 arbitrary unit (AU)/L at the basic level to (46.395±22.001)× 103 AU/L on the 12th week(F=14.642,t =-7.787,P＜0.05).On the 18th week serum T4,TRAb N and TRAb C decreased to (36.64±23.68) nmol/L,(0.094±0.053) U/L and (24.456±6.725)× 103 AU/L respectively,which were still higher than those preimmune levels(t=-4.161,-8.085,-9.008,all P＜0.05).There were no significant change of T4,TRAb N and TRAb C in the control group and blank group.After 4 times of immunization,the 99TcmO4-uptake by thyroids in immunized mice increased.The thyroid glands of immunized mice showed enlargement.Microscope examination showed that there were lymphocytes infiltration,colloid decrease and epithelial cell proliferation in thyroids of immunized mice.Conclusion GD mice models were successfully established by injecting recombinant plasmid pcDNA3.1/hTSHR268 and EP.