Gene Transfer And Expression Of Insulin Gene In Fibroblasts In Vitro

Objective Type 1 diabetes is a kind of auto-immune disease whose key feature is insulin-deficiency and hyperglycemia .There is no other treatment except for injecting ectogenesis insulin. Therefor, transgeneic insulin expression is one of the directions. Our research is to study human insulin gene transfer and expression in NIH3T3 cell by chitosan-mediated transfection.Methods An expression vector for human insulin gene was constructed by recombinant gene technique and introduced into NIH3T3 cells by chitosan-mediated DNA transfection .The transfection cells were grown in DMEM medium containing G418 at 72 hours after transfection,and the clones of cells were selected and continued to grow in G418 medium until the 21th day.To assay the transfected cells by immunohistochemical and insulin levels were monitored by ELASA.Results (1) Extracellular insulin levels in pCMV.Ins transfected group increased significantly as compared with untransfected group (P<0.01) and PCMV transfected group (P<0.01) .(2)there were no changes in extracellular insulin levels between untransfected group and pCMV.eGFP group (P<0.01) .Conclution (1)Human insulin gene was transfected successfully by chitosan-mediated and expressed efficiently in NIH3T3 cells,indicating the gene therapy of type 1 diabetes would be possible.(2)Having a lot of advantages ,chitosan would be a promising non-viral vector of the insulin gene.