The Mechanism Of Overexpression Of ICAM-1 Mesenchymal Stem Cells In Experimental Autoimmune Thyroiditis

Objective:1.Establishment of experimental autoimmune thyroiditis(EAT) model and give different mesenchymal stem cells therapy, and by detecting various index to observe therapeutic effect.2.using CM-Dil staining each group MSC to observe MSC distribution.3.extracting spleen cells protein, using western blot to detectet ERK and P38 pathways change.Methods:1.Purchased 60 6 week-old female C57BL/6 mice,feeded one week, established EAT animal models, divided into six groups,each group had 10 mice, in addition to the normal group, the other group mice were immunized by mixed well pig thyroid immunoglobulin(Porcine Thyroglobulin, PTg) with complete Freund’s adjuvant(Freund’s adjuvant complete, CFA) and incomplete Freund’s adjuvant(Freund’s adjuvant incomplete, IFA) at the 0th and 14 th day;the treatment group mice were given 3×105corresponding MSC therapy at the 0th and 14 th day,the normal group and the model group mice were injected of saline as control;at the 28 th day, weighed each group mice, picked out eyeball to collect blood, measured serum TT3, TT4, TSH,TGAb, TPOAb, TMAb level, separated thyroid tissue and HE stained to observe the severity of thyroid damage in each group, take out the mice spleen in each group and weighed, calculated spleen index(spleen weight/body weight),crushed the spleen,lysised red blood cell, washed,and got mononuclear cells;Then,added TRIzol reagent into the cells, extracted RNA, reversed transcriptase,used q-PCR to detect expression leves of inflammatory factors IL-4, IL-10, IL-17 and INF-γ.2.Purchased 60 6 week-old female C57BL/6 mice, used pig thyroid immunoglobulin with Freund’s adjuvant to establish EAT model, at the 0th and 14 th day,gave homologue treatment;but before gave treatment, MSC cells were stained by CM-Dil,then injected 3×105MSC per mice through intravenous;at the 28 th day,mice were anesthetized, perfused heart, collected thyroid gland and lung, made frozen section to observe the distribution of MSC in mice thyroid gland and lung.2.Toke the above mononuclear cells 5×106 per group, added the cell lysate to extracte cytoplasmic protein, used BCA to measure portein concentration, through western blot to detect the change of P38 and ERK of MAPK pathway.Results:1.The results of serum hormones and antibodies correlated with thyroid gland were showed that TPOAb、TGAb、TMAb、TSH levels in model group were higher than nomal group,were obviously higher than treatment group,but C3H10T1/2-ICAM-1/MSC group had the best effect and the difference was statistically significant(P<0.05).TT4 levels in model group were lower than nomal group,were obviously lower than treatment group,but C3H10T1/2-ICAM-1/MSC group had the best effect and the difference was statistically significant(P<0.05). HE pathology results showed thyroid folliculars were destructed,unequal in size,epithelial cell were hyperplased,inflammatory cells were infiltrated in model group. C3H10T1/2-ICAM-1/MSC group and primary MSC group thyroid damage were significantly reduced, inflammatory infiltration was reduced, but C3H10T1/2 / MSC group and C3H10T1/2-MIGR1/MSC groups’ therapeutic effect were not so good. spleen index in model group was significantly higher than normal group, C3H10T1/2-ICAM-1/MSC group spleen index was significantly improved than C3H10T1/2-MIGR1/MSC group, and the difference was statistically significant(P<0.01),spleen index in other treatment group were not improved so well. Inflammatory cytokines were detected in vivo and in vitro experiments by q-PCR.IL-10 and IL-4 levels in model group were lower than normal group, IL-17 and INF-γ were higher, compared with C3H10T1/2-MIGR1/MSC group,the levels of IL-10 and IL-4 were increased significantly in C3H10T1/2-ICAM-1/MSC group, the levels of IL-17 and INF-γ were significantly reduced, the difference was statistically significant(P <0.05).2.MSC homing studies have shown that C3H10T1/2-ICAM-1/MSC were homed to the thyroid gland most, primary MSC were mainly retented in the lungs.3.Western blot results showed p-ERK and p-P38 were enhanced in model group, were obvious decreased in C3H10T1/2-ICAM-1/MSC group.Conclusion:1No matter through pathology results, serum antibodies,inflammatory factors levels or spleen index levels, C3H10T1/2-ICAM-1/MSC group had the best therapeutic effect.2. MSC homing studies have shown that C3H10T1/2-ICAM-1/MSC were homed to the thyroid gland most,so ICAM-1 molecule could promote MSC homing3. C3H10T1/2-ICAM-1/MSC could inhibite ERK and P38 signaling pathways phosphorylation in mice immunocyte,regulate relevant gene experession in immunocyte, thus influence immunocyte differentiation, proliferation, immigration and so on.