Protective Effect Of Combined Intervention With Anti-CD20and Adenovirus Vector Mediated IL-10on Endogenous Islet β-cells In NOD Mice

Objective To investigate the protective effect of combined intervention with Anti-CD20and adenovirus vector mediated IL-10on islet B-cells in NOD mice.We demonstrate that combined immunotherapy can prevent or delay diabetes and promote B-cell growth and/or limit B-cells apoptosis.Methods Twenty-four female NOD mice (3～5weeks old) were randomly divided into four groups. Mouse①Anti-CD20.②Anti-CD20+IL-10、③Ad-mIL-10and④Normal Control(NC) groups were intraperitoneally injected of250ug Anti-CD20antibody, combined Anti-CD20antibody and0.1ml Ad-mIL-10,0.1ml Ad-mIL-10and0.1ml physiological saline, respectively. All mice were weekly monitored for body weight, urine glucose and blood glycose, and sacrified12weeks after injection. Their serum levels of Insulin, IL-10, CD20and CD19were measured and the degree of insulitis and the local expression of Insulin, Caspase3, IL-10and CD20gene were observed. Apoptosis was measured by using a TUNEL assay. Expreesions of Bcl-2, Caspase3and PDX-1were estimated by Western Blot and Real-time Polymerase Chain Reaction(RT-PCR).Results1.The body weight and blood glycose were both maintained to be normal in all groups;2.After combined Anti-CD20and IL-10intervention, the serum Insulin and IL-10levels were enhanced (vs Anti-CD20组、NC组,P=0.0002; vs IL-10组, P=0.01), and the serum CD20and CD19level were not enhanced or decreased compared with other3groups; Insulin, IL-10levels in pancreas also were enhanced;3. Compared with Normal Control group, insulitis severity (Anti-CD20组vs NC组P=0.0029; IL-10组vs NC组, P=0.0066) and B-cells apoptosis (NC组vs Anti-CD20, P=0.0001; NC组vs IL-10组,P=0.001)were decreased in Anti-CD20and IL-10groups; Lower insulitis score(P<0.0001)and β-cells apoptosis (vs IL-10组、NC组,P<0.0001; vs Anti-CD20组,P=0.001)compared to Anti-CD20, IL-10and NC groups were found in Anti-CD20+IL-10group;4. Caspase3level in pancreas was decreased in Anti-CD20, IL-10and Anti-CD20+IL-10groups; CD20levels in pancreas were enhanced in Anti-CD20+IL-10group; Decreased expression of Caspase3and enhanced expression of Bcl-2and PDX-1were observed in the combination group in pancreas.Conclusion The administration of Anti-CD20antibody and adenovirus vector mediated IL-10showed protective effects on islet β-cells in NOD mice, prevented diabetes development, reduced the β-cells apoptosis and insulitis, provided important guidance as a potential means of diabetes intervention.