Objective: 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 mellitus.
Methods: The recombinant adenovirus vector Ad-mIL-10 was constructed and transfected into RINm5F cells (Ad-mIL-10 group). Untransfected RINm5F cells and Ad-eGFP-transfected cells were used as controls. The expression of mIL-10 was examined by RT-PCR and Western blot. The levels of IL-10 in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). For a determination of insulin release, the cells were cultured with high glucose (16.7 mmol/L) for a 1-hour co-incubation. Then radioimmunoassay was used to detect the level of insulin in supernatant. After induction of IL-1beta, the levels of nitric oxide (NO) and nitric oxide synthase (NOS) were measured, the apoptosis of transfected cells was detected by Hoechst 33258 staining and Fas expression by flow cytometry.
Results: Both mRNA and protein of Ad-mIL-10 were successfully expressed in RINm5F cells. Expression of mIL-10 gene resulted in significant increases in insulin secretion in response to high glucose. Compared with uninfected control and Ad-eGFP infected group, Ad-mIL-10 infected group had decreased levels of NO and NOS induced by IL-1beta [NO level (nmol/10(6) cells): 52.9 +/- 3.2 vs. 227.3 +/- 26.4, 235.1 +/- 28.6, both P < 0.05; NOS level (U/10(6) cells): 9.3 +/- 1.2 vs. 29.8 +/- 2.5, 30.5 +/- 2.8, both P < 0.05]. Furthermore, Ad-mIL-10 gene transfer led to a profound reduction of Fas-expressing islet cells under the induction of IL-1beta. Fas-expressing islet cells in Ad-mIL-10 group were significantly lower than those in uninfected group and Ad-eGFP-transfected group (24.6% +/- 1.0% vs. 33.3% +/- 5.1%, 32.6% +/- 1.1%) (P < 0.05). The apoptotic rates of Ad-mIL-10 group were lower in comparison with the other two groups (9.4% +/- 1.1% vs. 19.2% +/- 2.2%, 20.6% +/- 2.3%, P < 0.05).
Conclusions: IL-10 gene transfer to islet beta cells may be beneficial in maintaining beta cells function, protecting islet cells from IL-1beta-mediated apoptosis and promoting islet cells survival. It is a potential therapy for type 1 diabetes mellitus.