| T1D is an autoimmune disease characterized by insufficient insulin secretion and progressive damage of islet b-cells.Because of the absolute lack of endogenous insulin,a systemic chronic metabolic disease characterized by hyperglycemia can cause three major metabolic disorders and water and electrolyte metabolic disorders.Long suffering patients often accompanied by cardiovascular and cerebrovascular diseases,kidneys,eyes and nerves and other pathological changes.Currently,insulin injection is still the main measures of treatment of this disease.However,insulin injection can't prevent apoptosis of pancreatic cells.Brittle diabetes severely affect patient's health and quality of life.It is showed that there are still residual islet beta cells in T1D patients'body.This type of Islet?-cells still have the ability to replicate and updates.Therefore,protection of these residual Islet?-cells'function is very important for reducing the incidence of T1D's acute complications,chronic complications and brittle diabetes[1].The immune imbalance theory is generally believed that the imbalance of Th1/Th2 cell subsets and excessive activation of B-lymphocytes are the pathogenic causes of T1D[2-4].IL-10 is an important cytokine secretion in Th2 cells.In this study,we successfully constructed adenovirus vector carrying rat IL-10 gene and confirmed the expression of exogenous IL-10 can correct the imbalance of the cell subsets ratio[5].CD20 is an importantcellular elements on surface of B cells.B cells can be selectively depleted using the anti-CD20 monoclonal antibody combining with CD20[6].In this study,combined applicaition of IL-10 and anti-CD20 monoclonal antibody in NOD mice with diabetes at early onset stage and was conducted.The combined applicaition's effects on blocking the Immune cells apoptosis of islet?cells and mediating proliferation of islet?cells was observed.Objective:To investigate the regenerative effect of combined Intervention with Anti-CD20 monoclone antibody and Adenovirus Vector Mediated IL-10?interleukin 10?on Endogenous Islet?-cells in nonobese diabetes?NOD?mice at early onset.Methods:24 female NOD mice at age of 17–20 weeks old and with Type 1 diabetes mellitus?T1D?diagnosed within 3 days were randomly divided into 4 groups.Mouse?1?Anti-CD20,individual therapy?2?CD20+IL-10,combination therapy?3?Ad-IL-10 and?4?Normal Saline?NS?groups were intravenously injected with 250ug Anti-CD20 antibody,mixture of 250ug Anti-CD20 and 0.1 ml Ad-mIL-10,0.1 ml Ad-mIL-10,0.1 ml NS,respectively.The total 4 injections was initiated per three days,with a double doses for the first time.All mice were closely monitored for blood glycose,and sacrificed 9 weeks after the last injection.Remove eyes of the mice,blood samples were gathered,centrifuged after standing.The upper Serum samples were detected with Enzyme-linked immunosorbent assay?ELISA?.Pancreases of the mice were dissected.Part of the pancreases were estimated by Real-time Polymerase Chain Reaction?RT-PCR?and Western Blot.The others were cut out to make paraffin block by 4%polyoxymethylene.The paraffin section was detected by immunohistochemistry and immunofluorescence.Result:Expression quantity of recombinant adenovirus fluorescence carrying green fluorescent protein and rat IL-10 gene was high in HEK293 cell.A significantly lower blood glycose levels was observed in NOD mice treated with combination therapy.The serum level of TNF-?and IFN-?were decreased and the level of TGF-?increased in combination therapy.The C peptide level of combination therapy group were elevated.Insulin expression of local pancreas was elevated.Immunohistochemistry showd that the Ngn3 level of combination group was decreased?p<0.05?.Immunofluorescence showd that the number of Pdx-1 and Co-expression insulin cells increased significantly.The qPCR results showd that Pdx-1?Pax4?Nkx6.1?Mafa gene expression of mouse pancreas in combination group rised significantly,versus the other groups?P<0.05?.Western blot results showd that increased expression of FPdx-1?Pax4?Nkx6.1?Mafa were observed in the combination group than Anti-CD20 group,IL-10 group and NS group?p<0.05?.Conclusion:Recombinant adenovirus-mediated IL-10 gene?Ad-IL-10?and null vector?Ad-GFP?are successfully enriched and amplified,which has established foundation for subsequent animal experiments.Adenovirus vector-mediated murine IL-10?Ad-mIL-10?shoed high gene expression in local pancreas.Adenovirus-mediated IL-10 gene in combination with anti-CD20 monoclonal antibody,can promote the secretion of insulin to NOD mice,blood glucose controlThe Insulin secretion of NOD mice can be promoted to control blood sugar by combining adenovirus vector-mediated murine IL-10 with Anti-CD20 antibody.Meanwile,the combined Intervention can promote the regeneration of Islet?-cells by activating Pdx-1-Ngn3-Pax4-Nkx2.2-Nkx6.1 pathway or promoting the expression of Mafa.It can provide a protective effect on residual Islet?-cells function. |
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