Establishment Of Graves Disease Mice Model And Experimental Study Of¹³¹I And Anti ICAM-1mAb Therapy

Objective:The Graves Disease (GD) BALB/c mice model was built. Prepare the monoclonal antibody against Intracellular adhesion molecule-1(ICAM-1). Study the GD therapeutic effect of131I and monoclonal antibody against ICAM-1(anti ICAM-1mAb).Methods:1. Build the GD BALB/c mice model. We injected recombinant plasmid pcDNA3.1/hTSHR268to mice gastrocnemius and used the technique of electroporation (EP) at the same time and the same location to enhance immunization. Analyse the content of thyroxine (T4), thyroid stimulating hormone receptor N-terminal antibody (TRAb N), thyroid stimulating hormone receptor C-terminal antibody (TRAb C). Perform radioactive isotopes99mTcO4-imaging and thyroid pathology analysis.2. Prepare the anti ICAM-1mAb. We used pure ICAM-1protein to immunize BALB/c mice and removed mice’s spleen, fused whose cells with myeloma cells (SP2/0). We selected the secration wells, sub-cloned them and prepared mAb in quantity by the method of inducing in vivo. And then, we acquired the anti ICAM-1mAb with biological activity by purified ascites.3. Treat the GD model mice experimentally by131I and anti ICAM-1mAb. We treated the GD model mice by intraperitoneal injection of131I and anti ICAM-1mAb respectively, and analysed the content of T4, TRAb N, TRAb C. Radioactive isotopes99mTcO4-imaging and thyroid pathology analysis were performed. At last, we compared the results above with control group and blank group to evaluate the GD therapeutic effect of131I and anti ICAM-1mAb.Results:1.1We built GD BALB/c mice model successfully by injection recombinant plasmid pcDNA3.1/hTSHR268to mice gastrocnemius and EP at the same time and the same location to enhance immunization, the positive rate of which was80%(24/30). 1.2We analysed the content of T4, TRAb N and TRAb C of24GD model mice. The results showed that all of them increased efficiently. After stopping the immunization, although the levels of T4, TRAb N and TRAb C decreased a little, they were still on the higher levels than preimmune (P<0.05). There were no obvious variation in the control group and blank group.1.3After4times of immunization, the ability of radioactive isotopes99mTcO4-uptake of immunized mice’s throid increased signifigantly. There were no obvious variation in the control group and blank group.1.4There were morphological changes of the thyroid glands of immunized mice, which were enlarged. Microscope examination of the thyroid tissues revealed that there were lymphocytes infiltration, less colloid and height of the epithelial cells increasing in mice immunized.2.1After3times immunization, the BALB/c mice were immunized successfully by pure ICAM-1pretein. The hybridoma cells were performed by fusing spleen cells of immunized mice and myeloma cells SP2/0.2.2There were174of192wells which were observed hybridoma cells. The fusion rate was90.6%. Thirteen of them were positive secretion, and the positive rate was7.5%. After screening again, there were3positive wells, which were C9, G10and H7. All of them were secreted antibody positively by twice sub-clone and identified as IgG1.2.3The titre of mAb induced by peritoneal fluid reached1:200000, which was only1:1000in liquid supernatant of hybridoma cells.2.4Protein quantitative standard curve was drawn according to the respective absorbance of standard protein concentration. The fomular was y=0.0014x+0.011. According to the curve, the protein concentrations of C9, G10and H7were917.8571μg/ml,650.0000μg/ml and767.8571μg/ml.3.1After the therapy of anti ICAM-1mAb, the levels of T4, TRAb N and TRAb C decreased differentially. The content of TRAb C was normal.3.2After the therapy of131I, all of the levels of T4, TRAb N and TRAb C decreased obviously. The content of T4was below the normal level. The mice were hypothyroidism. The contents of TRAb N and TRAb C were normal. 3.3The99mTcO4-uptake of anti ICAM-1mAb terapy group deceased, which was normal. And in131Ⅰ therapy group, it was below the normal level.3.4There were no pathological differences between different therapy groups and control group.Conclusion:1The method of immunized BALB/c mice by injection recombinant plasmid pcDNA3.1/hTSHR268and EP can build GD model successfully.2The technology of immunization and hybrid can prepare anti ICAM-1mAb successfully.3Both131Ⅰ and anti ICAM-1mAb may be used for treatment of GD model mice.