Study On The Abnormality Of The Mechanism Of Peripheral Immune Tolerance In NOD Mice

Autoimmune tolerance is physical status which immune system doesn't response to self antigens. Lots of researches suggest central as well as peripheral tolerance take effect. Most autoreactive cells with high affinity to self antigens can be clone deletion by negative selection, which called central tolerance. But it couldn't remove all the cells, and the survival will reach the peripheral tissues with potential pathogenicity. It needs peripheral mechanism to prevent us from immune response. Several ways can regulate these potential autoreactive lymphocytes, including clone anergy, clone deletion, immune deviation (balance of Th1/Th2), immune privilege and immune suppression/regulation.Type 1 diabetes mellitus is T lymphocytes mediated autoimmune disease. On the base of heritage, it is autoimmune response to islet beta cells with character of insulitis. Then the damage of islet cells will destroy insulin production and secretion and cause disorder of glucometabolism. Although the morbility increases in the world, the exact pathogenesis is unknown. At present, no approach can completely cure it. But the abnorbility of islet, disruption of immune tolerance and immune irregulation in diabetes animal models occur as well as susceptible population to T1DM. Study on the mechanisms of models will necessarily provide available plan to prevent and treat it.In this study we take the 10 week old NOD mice as the model toexplore the pathogenesis from peripheral tolerance, which may provide scientific evidence for the precaution and treatment of T1DM. It includes two parts:一,Estimation of T1DM1. Detection on blood and urine glucose In order to confirm if NOD mice have diabetes, we detect the blood glucose with glucose oxidase method and urine glucose with urine glucose test paper. Results showed that both of them have no difference with age matched Balb/c mice. These indicated NOD mice didn't appear symptoms of diabetes.2. Test 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. No difference between two groups.3. 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. Results showed the number and density of islet reduced in NOD mice, and lots of inflammatory cells infiltrated in it. These suggest NOD mice have insulitis before the significant symptom of diabetes.二,Study on the abnorbility of the mechanisms in peripheral tolerance:1. The proliferation ability of spleenocytes in vitro In order to study the cellular immune function in peripheral of NOD mice, we detect the polyclonal transformation ability of lymphocytes after stimulating with concanavalin A (5μg/ml). It showed that lowerability in NOD mice than in Balb/c mice (P<0.05). This result indicated there was polyclonal proliferation deficiency in peripheral mature T lymphocytes in NOD mice.2. The level of cytokine secreted by spleenocytes In order to further know the state of cellular immune function of T lymphocytes in NOD mice, and the immunological reason for pathogenesis of this disease. We measured the concentrations of IFN-γand IL-4 in suspension of spleenocytes activated with ConA. Results showed that the level of IFN-γincreased significantly (P<0.01), and IL-4 reduced (P<0.05). These indicated IFN-γmainly secreted by Th1 cells plays a critical role in the process of damage of isletβcells by the means of unbalance of Th1/Th2.3. The proportion of CD4+CD25+ Treg In order to estimate the ability of peripheral immune regulation in NOD mice, and explore the exact effect of CD4+CD25+Treg during the process of diabetes. We analyzed the percent of CD4+CD25+Treg with latent regulation ability among CD4+ T cells in spleen. Result showed higher proportion of CD4+CD25+Treg in NOD mice compared to Balb/c mice (P<0.05). It suggests there is deficiency in peripheral tolerance in NOD mice. The increase of CD4+CD25+Treg number suppresses the function of effective T cells which lead to no symptom in NOD mice.4. The expression of Foxp3 in spleenocytes Transcript factor Foxp3 can reflect the function state of CD4+CD25+Treg in certain degree. Therefore we detected the level of Foxp3 mRNA in peripheral spleenocytes in NOD mice by RT-PCR. Result showed that thelevel of Foxp3 mRNA has rising tendency in NOD mice compared to age matched Balb/c. It indicated the increasing number of CD4+CD25+Treg doesn't accompany with higher function.ConclusionsAbove all, there were no symptoms of diabetes and at the stage of prodiabetes in 10 week old NOD mice. But lots of inflammatory cells had infiltrated in islet. The peripheral tolerance had changed, including deficiency of spleenocytes proliferation, unbalance of Th1/Th2. But the relative increase of CD4+CD25+Treg compared to CD4+ T cells suppress the autoreactive T lymphocytes, which prevent NOD mice from diabetes.