Preparation Of Graves Disease With Graves Ophthalmopathy Model In BALB/c Mice

ObjectiveTo establish Graves disease?GD?mice model by multi-injection of thyrotropin receptor?TSHR?extracellular domain A subunit recombinant plasmid pcDNA3.1/TSHR268 into bilateral gastrocnemius muscle sites of BALB/c mice.Observed the occurrence characteristics of Graves ophthalmopathy?GO?and explore the disease features of GD with GO model in BALB/c mice.Methods1.Thyrotropin receptor?TSHR?extracellular domain A subunit recombinant plasmid pcDNA3.1/TSHR268 preserved in our laboratory were prepared in mass and identified by polymerase chain reaction?PCR?.2.In the model group,the above plasmid 50?g/50?L were injected into the bilateral gastrocnemius muscle of BALB/c mice immediately followed by electroporation to enhance the transfection effect.The control group mice were injected with sterile saline and electroporated,and the blank group mice were injected with sterile saline.All the mice were immunized for four times at 1st,4th,7th and 10th weeks,respectively.3.The serum thyroxine?T₄?,thyroid stimulating hormone?TSH?,thyrotropin receptor stimulating antibody?TSAb?and thyrotropin receptor stimulating blocking antibody?TSBAb?in mouse internal iliac vein were measured at the 0th,3rd,6th,9th,12th,15th,and 18th weeks.4.⁹⁹mTcO₄^-imaging and CT scan were taken for the mice to evaluate the thyroid uptake function and orbit extraocular muscle changes at 12th week.5.Thyroid-trachea preparations and the entire orbital bony tissues?including the orbital bones with the eyeball,extraocular muscles,and the optic nerve?were collected at the 18th weeks.Measure the size of the thyroid gland and the diameter of the eyeball and pathological changes of the thyroid and orbital tissue were evaluated by HE staining.Results1.After the second immunization,the levels of T₄ increased from 10.37±5.11ng/mL to 27.44±9.36 ng/mL in model groups,which were significantly higher than the control and blank groups?P<0.01?.The peak values were reached at 12th week and slightly decreased but remained higher than the other two groups until 18th week?P<0.01?.The level of TSH in the model groups decreased significantly from2.74±0.58?IU/mL before immunization to 1.88±0.65?IU/mL after the second immunization?P<0.01?and then decline.The level of TSAb in the model groups increased from 3.11±1.32?IU/mL to 98.46±13.12?IU/mL,TSBAb increased from5.06±2.71?IU/mL to 11.04±4.22?IU/mL.The TSAb and TSBAb in the model groups were significantly higher than those in the control groups and the blank groups?P<0.01?,and reached peak values at the 12th week and then decreased slightly.2.After four times of immunization,the thyroid uptake capacity of ⁹⁹mTcO₄^-in experimental groups were higher than those in the other two groups.The high resolution CT scan showed extraocular muscle thickening and spindle swelling in GD model mice and no abnormalities in tendon and muscle attachment points.The diameter of eyeball were no significant difference between each groups.3.The gross appearance of thyroid in the experimental groups were larger and fuller than thoset in the other two groups.HE staining showed a large number of lymphocytes infiltrated in the thyroid gland,the thyroid follicles were thinner and lighter color with some of the thyroid follicles hyperplasia and papillary folds.The above pathological changes were consistent with Graves'disease.None pathological changes were seen in the control groups and the blank groups.4.Hematoxylin-eosin staining of some mice in the GD model group revealed lymphocyte infiltration in the extraocular muscles and retrobulbar tissue,increased hyaluronan,adipose cell infiltration,and extraocular muscle thickening,which consistent with the pathological manifestations of GO.ConclusionsThe GD model can be successfully constructed by repeatedly injecting BALB/c mice with thyrotropin receptor extracellular domain A subunit recombinant plasmid pcDNA3.1/TSHR268 intramuscularly combined with electroporation.Some of these mice occur graves ophthalmopathy.