Studies Of Adenovirus-mediated Insulin-like Growth Factor 1 Gene In The Islet ¦Â Cell Damage Protection

PARTⅠ:ADENOVIRUS VECTOR MEDIATED RAT INSULIN-LIKE GROWTH FACTOR 1 GENE PROTECTS ISLETβCELL :RESEARCH IN VITROObjective To investigate the role of adenovirus vector mediated rat insulin-like growth factor 1 (rIGF-1 )gene on streptozotocin-induced isletβ-cells impairment in vitro.Methods Recombinant adenovirus encoding rIGF-1 gene was constructed, and then infected to RINm5F cells. RIGF-1 protein was detected by Western blot analysis and ELISA method. Then streptozotocin was used to induce RINm5F cells impairment. The levels of nitric oxide were detected in cells culture supernatants. The cells function was evaluated by glucose-stimulated insulin production. The apoptosis was analyzed by flow cytometry. Thiaoollyl blue viability assay was applied to exam the number of viable cells.Results The recombined adenovirus-rIGF-1 was constructed successfully and its titer was about 4.0×10~8 pfu/ml. The rIGF-1 was expressed in the RINm5F cells and cells culture supernatants. RIGF-1 can inhibite islet cell apoptosis and significantly decrease the level of NO induced by streptozotocin, while significantly increase insulin secretion and cells viability.Conclusion These results suggested that locally produced IGF-1 from cultured islets may be beneficial in maintainingβcells function, protecting islet cells from apoptosis -mediated factors and promoting islet survival, which showed the potential therapy for type 1 diabetes mellitus. The apoptosis induced by streptozotocin maybe NO-dependent. PARTⅡ; ADENOVIRUS VECTOR MEDIATED INSULIN-LIKE GROWTH FACTOR 1 GENE PREVENTS CYCLOPHOSPHAMIDE INDUCED DIABETIC MELLITUS OF NOD MICE: RESEARCH IN VIVOObjective : to investigate the protective role of adenovirus vector mediated rat insunlin-like growth factor 1(IGF-1) on cyclophosphamide-induced diabetic mellitus of non obesity diabetic mice (nod mice).Method : Twenty-four female nod mice aged four weeks were randomly divided into three groups. Group 1 received the intreperitoneal injection of saline(0.1ml), Group 2 received the introperitoneal injection of adenovirus vector mediated IGF-1 gene (0.1ml). Group 3 received the intropeirtoneal injection of adenovirus vector mediated empty green fluorescence protein(Ad-eGFP).All the mice were administered the introperitoneal injection of Cyclophosphamide (300mg/kg) to accelerate the progress of diabetic mellitus at the age of 5 weeks. We measure the weight and the blood sugar level weekly since at the age of 4 weeks. All the mice were killed when they developed diatetic mellitus or 30 days after our examination. We observed the incidence of diabetic mellitus ,the degree of pancreatis and the expression of IGF-1 detected with the immunohistochemical technique.Results: The weight in the Ad-IGF-1 gene group was higher than in the Ad-eGFP group and the saline group. The blood sugar lever in the Ad-IGF-1 group was lower than in the Ad-eGFP group and the saline group,however there was no statistical difference. Intraperitoneal injection of Ad-IGF-1 gene to NOD mice did not reduce the incidence of diabetes. The insulitis score and the severity of insulitis were not lower in Ad-IGF-1 gene group than in saline group .There were no statistical differences in the IGF-1 level in these three groups.Conclusion The Ad-IGF-1 gene did not protect nod mice from developing diabetic mellitus in our examination.