Study On The Relationship Between CD4~+/CD8~+ T Subsets, T-bet/GATA3 Gene Expression And Type 1 Diabetes Mellitus

Part one:Change and analysis on CD4+/CD8+ T substes in STZ-induced type 1 diabetic murine modelObjective: To study the changes of CD4+/CD8+ T subsets in STZ-induced type 1 diabetic murine model for explaining the mechanisms underlying type 1 diabetes mellitus (T1DM).Methods: STZ-induced type 1 diabetic murine model was established by intraperitoneal injection of 40mg/kg streptozotocin (STZ) for 5 consecutive days. FACS was used to determine the percentages of CD4+T cells, CD8+T cells and CD4+CD25+T cells in spleens, with MTT for lymphocyte proliferation, ELISA for cytokine (IL-2, IL-4, IL-10 and IFN-γ) levels in serum and HE staining for pancreatic pathological changes.Results: The markedly reduced CD4+T cells and CD8+T cells were detected in the STZ model mice at 2wk (P<0.05), with no significant change in CD4+/CD8+ ratio and CD4+CD25+T cells (P>0.05). At 4wk, CD4+T cells and CD4+/CD8+ ratio were shown to increase obviously (P<0.05, P<0.01), whereas CD8+T cells and CD4+CD25+T cells significantly reduced in the STZ model mice (P<0.05). The increasing of lymphocyte proliferation was observed in splenocytes of STZ model mice at both 2wk and 4wk (P<0.05). And high amounts of IFN-γwere measured in serum of STZ-diabetic mice at 2wk and 4wk (P<0.05, P<0.01), whereas levels of IL-2, IL-4 and IL-10 showed no significant difference compared to normal control (P>0.05). Inflammatory cells infiltrating in pancreatic islets were observed in pancreas of STZ model mice at 2wk and 4wk, with atrophied islets just seen in STZ-diabetic mice at 4 wk.Conclusion: There still existed the imbalance of CD4+/CD8+ T cells in the whole process of type 1 diabetes mellitus. The abnormal activation of CD4+Th1 cells and loss of CD8+T cells play important roles in the pathogenesis of T1DM.Part two: Contribution of diabetogenic CD4+ T cells in the Pathogenesis of Type 1 Diabetes Mellitus Objective: To investigate the role of diabetogenic CD4+T cells in the pathogenesis of type 1 diabetes mellitus (T1DM) by adoptive transfer of purified diabetogenic CD4+T cells.Methods: Autoimmune diabetes mellitus was developed by intraperitoneal injection of 40mg/kg streptozotocin (STZ) daily for 5 consecutive days in BALB/c mice as sources of donor cells. Spleen cells from diabetic mice were then cultured for 7 days in the stimulation of interleukin-2 (IL-2) to harvest diabetogenic splenocytes and purified diabetogenic CD4+T cells, which were subsequently adoptive transferred into BALB/c mice recipients with STZ twice. FACS, MTT, ELISA and HE staining were used to analyze the phenotypes of diabetogenic splenocytes, the lymphocyte proliferation, cytokine (IL-2, interferon-γ, IL-4, and IL-10) levels and pathological changes in pancreatic islets.Results: As few as 3×106 diabetogenic splenocytes successfully induced hyperglycemia and insulitis in recipients pretreated with STZ twice, whereas transfer of equal amount of purified diabetogenic CD4+ T cells alone failed to initiate hyperglycemia in STZ-twice recipients. FACS assay showed that T cells and CD4+T cells occupied 85.6% and 81.7% respectively in the diabetogenic splenocytes. MTT assay dispayed an obviously higher lymphocyte proliferation in both the diabetogenic splenocytes and purified diabetogenic CD4+T cells alone (P<0.05). A markedly enhanced lymphocyte proliferation was also seen in spleens of recipients receiving diabetogenic splenocytes transfer (P<0.05), with no significant difference for those injected with purified diabetogenic CD4+T cells alone (P>0.05). High amounts of IFN-γand IL-2 were observed in supernatants of both diabetogenic splenocytes and purified diabetogenic CD4+T cells (P<0.05). In addition, high level of IFN-γwas shown to exsit in serum of recipient mice receiving diabetogenic splenocytes transfer(P<0.01), whereas the contents of cytokines IL-2, IL-4 and IL-10 had no marked changes in comparison to normal control (P>0.05). Oppositely, a similar cytokines secretion profile as normal control mice was measured in serum of recipients with purified diabetogenic CD4+T cells transfer. Numerous lymphocytes infiltrating in pancreatic islets and severe isletβcell lesion were observed in pancreas of mice receiving diabetogenic splenocytes transfer by HE staining assay, and obvious inflammatory infiltrates were also found in pancreas of recipients injected with purified diabetogenic CD4+ T cells alone.Conclusion: The fact that the transfer of purified diabetogenic CD4+ T cells alone induced insulitis but no hyperglycemia in recipients shows that diabetogenic CD4+ T cells play the critical role in the initial stage of type 1 diabetes mellitus by priming inflammatory lymphocyte infiltrating in pancreatic islets rathehr than killing effect onβcells.Part three:Study on the relationship between mRNA expression of transcription factor T-bet/GATA-3 and type 1 diabetes mellitusObjective: To study the relationship between the expression of T-bet/GATA-3 mRNA and Th1/Th2 cytokines in type 1 diabetic murine model for revealing the differentiation of Th1/Th2 in type 1 diabetes mellitus (T1DM) on the level of transcription and investigating the invovment of T-bet/GATA-3 in the pathogenesis of T1DM.Methods: STZ-induced T1DM murine model was established by intraperitoneal injection of 40mg/kg streptozotocin (STZ) daily for 5 consecutive days in BALB/c mice and diabetic mice were sacrificed at 4wk post to last dose of STZ for experiments. Non-obese diabetic (NOD) mice were feeded up to 22wk in the specific-pathogen free (SPF) environment and diabetic NOD mice were randomly selected for assays. Total RNA was extracted by Trizol agent and cDNA was prepared using reverse transcription reaction (RT). Polymerase chain reaction (PCR) was used to examine the gene expression of transcription factors T-bet/GATA-3 and Th1/Th2 type cytokines IFN-γ/IL-4 in spleens, with ELISA for levels of cytokine IFN-γ, IL-4 in serum.Results: The increase of T-bet and IFN-γmRNA expression as well as the depression of GATA-3 mRNA was found in both the STZ-diabetic mice and diabetic NOD mice, whereas the expression of IL-4 mRNA was still not detected in any mice. The content of IFN-γsecretion in serum was significantly higher in STZ-diabetic mice and NOD diabetic mice in comparison to controls, wherea IL-4 levels showed no statistically reduction.Conclusion: Expression of transcription factor T-bet/GATA-3 mRNA in spleens was shown to correspond to levels of Th1/Th2 type cytokines in serum when T1DM in our experiments, suggesting that transcription factor T-bet/GATA-3 is associated with the onset of type 1 diabetes mellitus by affecting the differentiation of Th1/Th2 cells.