Establishment Of Graves Disease With Graves Ophthalmopathy Model In BALB/c Mice And Exploration Of New Therapy

Objective:To establish Graves disease?GD?with Graves ophthalmopathy?GO?model in BALB/c mice and explore the treatment effect using siRNA and monoclonal antibody against ICAM-1?anti ICAM-1 mAb?.Methods:1.To establish GD with GO model in BALB/c mice.Recombinant pcDNA3.1/TSHR289 plasmid was injected into BALB/C mice bilateral gastrocnemius muscle and electroporated at the same time.After three weeks,Measured the serum thyroxine?T₄?,thyroid stimulating hormone?TSH?,thyrotropin receptor stimulating antibody?TSAb?and thyrotropin receptor stimulating blocking antibody?TSBAb?.Use ^99mTcO₄^-imaging and pathology analysis to evaluate the changes of the thyroid uptake function and orbit extraocular muscle in the mice.2.The GD with GO model was successfully treated by siRNA and anti ICAM-1mAb.TSHR was used as a target to screen and design synthetic siRNA,transfected Nthy-ori-3-1 cells.Western blotting was used to detect the difference in expression levels of TSHR before and after transfection.Detect T₄,TSH,TSAb and TSBAb.Use^99mTcO₄^-imaging and pathology analysis to evaluate the changes after treated by siRNA and anti ICAM-1mAb.Results:?1?The GD with GO animal model was successfully constructed by injecting the recombinant plasmid pcDNA3.1/TSHR289 into the gastrocnemius muscle of BALB/c mice and electroporating at the same site.The experimental group was immunized serum T₄ increased from 12.74±4.14ng/mL to 70.84±16.46ng/mL,TSAb increased from 3.11±1.32?IU/mL to 483.48±44.29?IU/mL,TSBAb increased from4.24±1.10?IU/mL to 16.81±6.05?IU/mL.The levels were significantly higher than those in the control group and the blank group?P<0.05?.The serum TSH of the experimental group was significantly decreased from 2.63±0.55?IU/ml to0.05±0.02?IU/ml?P<0.05?,that was significantly lower than the other two group?P<0.05?.After immunization,the weight growth rate of the experimental group was significantly slower than that other two group.In the experimental group,the thyroid tissue was fuller than the other two groups.The thyroid ^99mTcO₄^-uptake ability was significantly higher than the other two groups.HE staining showed a decrease in thyroid follicular gelatin,showing no glial cavity,lymphocyte infiltration,and partial follicular cell proliferation.HE staining of heart tissue showed a right subepicardial calcium salt lesion occasionally.There was no significant difference between the eyes.Dyeing showed lymphocytic infiltration of the extraocular muscles and the posterior globules,infiltration of fat cells,and occasionally thickening of the extraocular muscles.?2?Western blotting results showed that the expression of TSHR was significantly decreased in Nthy-ori-3-1 cells transfected with siRNA.The serum T₄,TSAb and TSBAb of the mice treated were significantly lower than those before treatment,and significantly lower than the untreated control group,but still higher than the blank group.TSH increased significantly compared with before treatment,and was significantly higher than the untreated control group,but still lower than the blank group.After treatment,the body weight of the mice was increased compared with the control group but slightly lower than the blank group.After treatment,the thyroid ^99mTcO₄^-uptake ability decreased compared with the untreated control group,but it was still slightly higher than the blank group.There was no significant difference in the visual diameter and pathology of the mouse eyeballs after treatment compared with the untreated group.Conclusion:The recombinant plasmid pcDNA3.1/TSHR289 was injected into the gastrocnemius muscle of BALB/c mice and electroporated at the same site to successfully construct BALB/c mouse GD with GO animal model,siRNA and anti ICAM-1 mAb have a certain therapeutic effect for GD with GO model mice.