Study On The Dysfunction Of Central Immune Tolerance In NOD Mice

Autoimmune Disease (AD) is caused by the loss of self-tolerance. The development of AD reflected the deficient of the central tolerance and/or peripheral tolerance. The central tolerance was the results of the normal selection of T and B cells in central immune organs. The promiscuous expression of self-antigen in thymus is related with the consequence of T cells negative selection and is crucial for maintaining the intact of central tolerance. Type 1 diabetes (T1D) is a kind of autoimmune diseases, which the insulin-producing c?ells of the islets are destroyed by the immune system and leads to a lifelong depended with exogenous insulin. So far, the incidence of diabetes rises year by year and there was a tendency to younger population.During the past several decades, with the help of various diabetes animal models, we have got some knowledge about the pathogenesis of type 1 diabetes and more strategies to cure it. However, there are still some unsatisfied.In this study, we take the spontaneous diabetes model that is NOD mice as the studying object, profoundly explore the effect of central tolerance on the occurrence of diabetes by investigating the dysfunction of central tolerance in models. We researched from three parts:一.The diabetes associated indexes in NOD mice:1. Detection of blood and urine glucose: In order to judge if the NOD mice had diabetes mellitus, we tested the blood glucose with glucose oxidase method and urine glucose with urine glucose test paper. Results showed no difference between NOD mice with age matched Balb/c mice. Theseindicate NOD mice didn't appear symptoms of diabetes.2. Observation of pancreatic histology: In order to know the pathology changes of pancreas in NOD mice, we observe the histology changes of islet under light microscope after HE staining. Results showed that in NOD mice pancreatic islets were decreased in amount, rarely distributed, and low in density. Pancreatic islet cells decreased, with large amount of inflammatory cells infiltrating. These indicate the insulitis appeared before the obvious diabetes syndrome in NOD mice.3. Detection of the insulin autoantibody in serum: In order to know the state of humoral immunity, we tested the level of insulin autoantibody in serum with indirect ELISA. The result showed no difference between two groups. It indicates the normal humoral immunity at this stage of NOD mice.二.The immune function of thymus in NOD mice1. Observation of the weight and histology of thymus: In order to know the general morphous and construction of thymus, we weighed the thymus with analytic balance and calculated the thymus index. Also observed the histology of thymus under light microscope after HE staining. Results showed that the cortico-medullary junction was vague and cells in medulla were reduced but no obvious atrophy in thymus.2. Detection of the T lymphocytes function in thymus: In order to know the immune function of thymocytes, we detected with lymphocytes transformation assay with MTT method. Results showed that the thymocytes proliferation to ConA there were no significance compared with Balb/c control group. It indicates normal polyclonal proliferationof T lymphocytes in NOD mice thymus.3. Test of the level of IL-4 and IFN-γin suspension of thymocytes: In order to further know the immune function of T cells, we test the production of IL-4 and IFN-γby sandwich ELISA. Results showed that the level of IL-4 was lower in NOD mice than those in Balb/c group, but no significant difference with IFN-γ. These indicate the abnormal of Th cell differentiation in NOD mice thymus and lead to the unbalance of Th1/Th2.三.The state of central immune tolerance in NOD mice1. The expressions of promiscuous genes in thymus: In order to study if the changes of thymic construction can affect the ability of promiscuous gene expressed on TEC, we detected the mRNA level of diabetes associated antigen-Insulin and GAD67, diabetes unrelated antigen-PLP in thymus by RT-PCR. Results demonstrated that in NOD mice the level of Insulin expression in thymus were lower than those in Balb/c group, and GAD67 and PLP expression were no significance difference in two groups. These suggest that the absence expression of diabetes related gene in NOD mice thymus may induced abnormity of negative selection.2. The level of MHC-II on thymic epithelial cells: In order to know the level of MHC-II molecules in thymic epithelial cells, we detected it with FACS. Results showed it obviously decreased in NOD mice. It suggests the low level of MHC-II can influent the ability of epithelial cells to bind all kinds of peptides, which may affect the negative selection.3. The proportion of CD4+CD25+ cells among CD4+ T cells: In order to further study the composition of thymocytes. We analyzed the proportion of CD4+CD25+ cells among CD4+ T cells in thymus. Results showed that the percent of CD4+CD25+ T cells showed no difference in two groups. It indicates the production of CD4+CD25+Treg cells essential for peripheral immune tolerance unchanged in NOD mice.Conclusions1. NOD mice were at the stage of prone diabetes without general diabetes syndromes. But there have been inflammatory cells infiltration in pancreatic islet.2. The structural abnormalities and reduced number of epithelial cell in NOD thymus, addition with the decreased level of diabetes related promiscuous gene–insulin will lead to the abnormal negative selection to the autoreactive thymocytes. The level of MHC-II molecule on thymic stromal cells may induce the low ability binding all kinds of peptides, which can further affect negative selection. Above all, the deficiency of central tolerance in NOD mice may be the latent motivation for autoimmune diabetes.