| Objective: To investigate the possible role of AIRE in the development of thymic T cells and the pathogenesis of T1 D.We investigated the changes of AIRE,INS2 and GAD67 genes expression levels in the thymus tissue of normal mice and NOD/Ltj mice at different ages,and during the differentiation of m ESCs-DE-TEPs in vitro.Methods: 1)Thymus tissues of C57BL/6,BALB/c,NOD/Ltj mice at different ages were studied.The distribution and morphological characteristics of AIRE gene in thymus of mice were observed by K5/AIRE immunofluorescence staining.Western Blotting(WB)was used to observe the expression of AIRE in thymus tissue at different ages.The expressions of AIRE,INS2 and GAD67 genes in thymus tissue of C57BL/6,BALB/c and NOD/Ltj mice at different ages were observed by real-time quantitative fluorescence PCR(q PCR).2)Mouse embryonic stem cells(m ESCs)were induced in vitro to differentiate into endoderm(DE)and then to differentiate into thymic epithelial progenitor cells(TEPs).WB observed the expression change of AIRE during mescs-de-teps differentiation.q PCR was used to observe the expression changes of AIRE gene,INS2 gene and GAD67 gene in m ESCs-DE-TEPs differentiation.Results: 1)Histological localization of AIRE: immunofluorescence staining results showed that AIRE was spot-like in the thymus of mice,mainly distributed in the area of medullary thymus epithelial cells with positive K5.2)Aging changes(1)q PCR results showed that the expressions of AIRE,INS2 and GAD67 in thymus tissues of C57BL/6 and BALB/c mice with 2W,16 W and 32 W gradually decreased,with statistically significant differences(p<0.05);(2)WB results showed that the expression of AIRE in thymus tissue of C57BL/6 mice decreased gradually in2 W,16W,32 W and 48 W,and the difference was statistically significant(p<0.05).3)The relationship between AIRE and the type 1 diabetes(1)q PCR results showed that AIRE,INS2 and GAD67 genes in NOD/Ltj mice decreased with theincrease of age,and the differences were statistically significant(p<0.05);In NOD/Ltj mice of the same age,the expression levels of INS2 gene and GAD67 gene were decreased in the hyperglycemia group compared with the normal group,and the differences were statistically significant(p<0.05).(2)WB results showed that AIRE protein expression in NOD/Ltj mice decreased with the increase of age,and the difference was statistically significant(p<0.05);In the same week-old NOD/Ltj mice,AIRE protein expression in the hyperglycemia group was not significantly different with the normal group..4)AIRE gene expression during m ESC differentiation to TEPs(1)q PCR results showed that relative expressions of AIRE,INS2 and GAD67 genes all increased during m ESCs-DE-TEPs differentiation,with statistically significant differences(p<0.05).(2)WB results showed that AIRE protein expression decreased during m ESCs-DE-TEPs differentiation,and the difference was statistically significant(p<0.05).Conclusion: 1.The expression of AIRE in thymus tissue of C57BL/6 and BALB/c mice was Age-related and correlated with INS2 gene and GAD67 gene changes,which may be related to the maturation and development of T cells in thymus.2.The synergistic changes of AIRE and INS2 gene and GAD67 gene related to Type 1 Diabetes may be related to the progression of autoimmune Type 1Diabetes in NOD/Ltj mice.3.During the differentiation of m ESCs-DE-TEPs in vitro,the synergistic changes of AIRE,INS2 and GAD67 may be an important factor in promoting the maturation and development of T cells after transplantation in vivo. |
