| Objective The experimental autoimmune thyroiditiswas treated with mesenchymal stem cells(MSCs)in C57 BL / 6 mice,and the mechanism of MSCs in the treatment of EAT was elucidated.The aim of this research is to provide clinical application of MSCs and theoretical and experimental basis of autoimmune thyroiditis(AIT).Methods1.Establishment of mouse EAT model.On day 1 and day 14,female C57 BL / 6 mice were given porcine thyroglobulin and complete Freund’s adjuvant mixed solution and porcine thyroglobulin mixed with incomplete Freund’s adjuvant solution.The expressions in serum of thyroid autoimmune antibodies were detected by enzyme-linked immunosorbent assay(ELISA).And thyroid histopathology,spleen index were detected to testify whether the model were successfully established.2.EAT model mice were treated with MSCs.MSCs were isolated from bone marrow of one-week-old mice,and the morphology of cells was observed under inverted microscope.The cell phenotype of MSCs was detected by flow cytometry.After successfully established the EAT model,MSCs were transplanted on day 0 and day 14 respectively.Comparing the changes of the spleen index,the changes of the thyroid autoantibody TPOAb,TgAb and TMAb,the HE staining results of the thyroid gland in each group,and the changes of spleen cell immune function in EAT model group and MSCs-treated group detected by flow cytometry,to testify the therapeutic effect of MSCs on EAT mice.3.Study on the Mechanism of MSCs in treating EAT.The apoptosis of thyroid tissue in EAT mice was detected by immunohistochemistry and immunofluorescence,The expression of apoptotic factors in normal control group,EAT model group,MSCs treatment group and MSCs was detected by Q-PCR.MSCs were transfused with CM-Dil labeled cells in EAT model mice,and the homing ability of MSCs was detected by immunohistochemical staining.Experiment in vitro to study the mechanism of MSCs protective effect on thyroid tissue apoptosis in EAT mice.Results1.The mouse EAT model was successfully established by subcutaneous injection of porcine thyroglobulin and two Freund’s adjuvant.2.The results of flow cytometry showed that MSCs were highly expressed in stem cells.After treatment with MSCs,histopathological HE staining showed that the thyroid tissue of MSCs treatment group had different degree of thyroid follicle destruction and lymphocyte infiltration,but compared with the EAT model group,the lesion was mild,And no obvious damage to the follicle.There was no obvious difference in spleen index among three groups.The results of ELISA showed that the expression of thyroid microsomal antibody,thyroid peroxidase antibody and thyroglobulin antibody in MSCs treatment group were significantly lower than those in EAT model mice.3.Immunohistochemistry and immunofluorescence staining showed that the expression of Caspase3 in the thyroid tissue of the mice treated with MSCs was significantly lower than that of the EAT model group in the EAT model group,The Q-PCR results showed that a large number of FasLs were expressed in the EAT model group.The expression of apoptotic receptor Fas in thyroid tissue was significantly higher in MSCs and normal controls.Fluorescence tracing results showed that MSCs could be homing to the thyroid tissue of inflammatory.In vitro co-culture results confirmed that EAT model mice splenic lymphocytes can act on normal thyroid tissue,induced apoptosis factors Caspase3,Caspase6 and Bcl-2 high expression,triggering apoptosis,and MSCs can effectively prevent EAT model mice Effects of splenic lymphocytes on the pro-apoptotic effect of thyroid tissue in normal mice.ConclusionMSCs can effectively hominate into the thyroid tissue of inflammation.By expressing Fas receptor,the killing effect and apoptosis of thyroid tissue of CD8 + lymphocytes in EAT model mice are prevented,and the thyroid tissue cells are protected and their own Immune disorders caused by thyroid injury,thereby delaying the progress of experimental autoimmune thyroiditis. |
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