Combination Therapy With IGF-1 Gene And IL-10 Gene Prevents Type 1 Diabetes In NOD Mice

Part IObjective To evaluate the efficacy of adenovirus vector-mediated murine interleukin-10 (mIL-10) gene transfer to rat beta cell-RINm5F cells in vitro and to explore the potential value of gene therapy in type 1 diabetes.Methods The recombinant adenovirus vector, Ad-IL-10 was constructed and transduced into RINm5F cells. The expression of mIL-10 was checked by RT-PCR and Western blot. The levels of IL-10 in supernatant were checked by enzyme-linked immunosorbent assay (ELISA). For determination of insulin release the cells (Ad-IL-10-transfected cells, Ad-GFP-transfected cells, and nontransfected cells) were cultured with high glucose (16.7mmol/L), after 1h co-incubation radioimmunoassay was used to detect the level of insulin in the supernatant. After induced by IL-1β, the levels of nitric oxide (NO) and nitric oxide synthase (NOS) were measured, apoptosis of transfected cells was detected by Hoechst 33258 staining and Fas expression by Flow cytometry method.Results Ad-IL-10 was successfully expressed of both mRNA and protein in RINm5F cells. Expression of the mIL-10 gene resulted in significantly increased in insulin secretion in response to high glucose. Compared with uninfected control and Ad-GFP infected group, Ad-IL-10 infected group decreased the level of NO and NOS induced by IL-1β. Furthermore, Ad-IL-10 gene transfer led to a profound reduction of Fas-expressing islet cells which was induced by IL-1βand the apoptotic rates of Ad-IL-10 group were decreased versus the other two groups(P<0.05).Conclusions These results suggested that IL-10 gene transfer to islet beta cells may be beneficial in maintaining beta cells function, protecting islet cells from apoptosis-mediated by IL-1βand promoting islet cells survival, which showed the potential therapy for type 1 diabetes mellitus. PartⅡObjective In this study, we investigated whether IL-10 combined with IGF-1 gene transfer could 1) prevent autoimmune destruction of islet beta-cells 2) promote beta-cell growth and/or limit beta-cell apoptosis.Methods We constructed an adenoviral vector containing IL-10 gene (Ad-IL-10) or IGF-1 gene (Ad-IGF-1) separately. Female NOD mice at 9 weeks of age were treated with Ad-IL-10 and/or Ad-IGF-1, and observed up to 30 weeks of age. Blood glucose concentration was measured weekly. Serum insulin, cytokine production were tested. Pancreatic histology was measured for determination of insulitis grades. Pancreatic insulin content and beta-cell mass, proliferation were measured. Apoptosis was measured by using a TUNEL assay. Expressions of Fas and caspase-3 were estimated by immunohistochemistry analysis.Results A significantly lower diabetes incidence (p<0.01) was observed in NOD mice treated with Ad-IL-10+Ad-IGF-1 and Ad-IL-10 alone or Ad-IGF-1 alone compared to mice treated with Ad-GFP or saline alone. Lower insulitis score compared to control mice was found in Ad-IL-10+Ad-IGF-1 group (p<0.01). The combination therapy increased pancreatic insulin content and beta-cell mass, and beta-cell apoptosis was decreased. Decreased expression of Fas and caspase-3 were observed in the combination group.Conclusions These results present a new treatment for type 1 diabetes mellitus therapy. IL-10 and IGF-1 gene therapies are able to prevent and delay diabetes development, reduce autoimmunity and increases pancreatic beta-cell mass, indicating promising potential of these therapies as a new treatment strategy for type 1 diabetes mellitus.