hTSHR188-403bp基因免疫BALB/c小鼠建立Graves’病动物模型

Genetic immunization of BALB/c mice with extracellular domains of human TSH receptor provides a model of Graves' disease

目的:构建pcDNA3.1/hTSHR(188～403bp)重组质粒,并将其基因免疫BALB/c小鼠建立Graves’病动物模型。方法:RT-PCR扩增hTSHR膜外区188～403bp,与pcDNA3.1载体连接,构建重组质粒pcDNA3.1/hTSHR,用酶切、PCR及测序方法进行鉴定。于1、4、7、9、10周将重组体免疫实验组小鼠,对照组注射生理盐水,0、6、11周取血,检测血清TRAb、T4、TSAb、TBAb。取甲状腺常规病理切片,HE染色镜检。结果:构建的重组质粒经酶切、PCR鉴定证明插入方向正确,测序结果与GeneBank中hTSHR的相应片段序列相同。免疫组T4、TRAb和TSAb水平均从第6周开始显著升高(P<0.05),TBAb差异无统计学意义(P>0.05),对照组差异均无统计学意义(P>0.05)。实验组甲状腺病理检查结果,显示滤泡增生,胶质浓缩等Graves’病的病理表现,对照组形态正常。结论:构建了重组质粒pcDNA3.1/hTSHR(188～403bp),成功建立了类Graves’病动物模型。

Objective：To construct the recombinant plasmid pcDNA3.1/hTSHR（188～403 bp）and establish the animal models of Graves＇ disease by genetic immunization.Methods：hTSHR was obtained through RT-PCR and linked with pcDNA3.1.Constructed pcDNA3.1/hTSHR was identified by restricting enzyme digestion analysis,PCR amplifying and DNA sequencing.BALB/c mice were immunized on weeks 1,4,7,9 and 10 with recombinant plasmids.Control group were injected with normal sodium.Animals were bled at weeks 0,6 and 11 and sacrificed at week 11 for thyroid histology.Histological analysis of thyroid tissue was carried out with HE staining.The level of TRAb in sera was assayed by ELISA.The level of T4,TSAb,TBAb was determined by radioimmunoassary（RIA）.Results：Restriction enzyme digestion,PCR amplifying and DNA sequencing confirmed that pcDNA3.1/hTSHR had been constructed successfully.In the mice immunized with pcDNA3.1/hTSHR,sera T4,TRAb and TSAb elevated significantly（P〈0.05）,TBAb has no statistically significance（P〉0.05）.A great number of new follicles,concentrated colloid and follicular epithelial cells hyperplasia were observed in Thyroid tissue.Sera T4,TRAb,TSAb and TBAb of control group showed no statistically significance（P〉0.05）.Conclusion：The pcDNA3.1/hTSHR has been successfully constructed.Animal models of Graves＇ disease have been made successfully by genetic immunization.