Co-transfection of GK and mhPINS genes into HepG2 cells confers glucose-stimulated insulin secretion.

BackgroundThe purpose of this study was to construct an ‘artificial beta cell’ that can exhibit physiologic glucose-stimulated insulin secretion for the treatment of type 1 diabetes.MethodsRetroviral vector containing the glucokinase (GK) gene and mutated human proinsulin (mhPINS) gene was constructed. HepG2 cells were first infected with recombinant retrovirus carrying the GK and mhPINS genes, then selectively cultured with G418 to obtain the positive clones. GK and mhPINS gene transcription and expression were identified by radioimmunity, Western blot and RT-PCR techniques. Finally, the dose–response effect of glucose on insulin secretion from those HepG2 cells that expressed both GK and mhPINS genes was tested with HepG2 cells that only expressed the mhPINS gene as a control.ResultsHepG2 cells with transferred GK and mhPINS genes were selectively cultured with G418 and the positive clones were obtained in 3 weeks. Four clones with GK and mhPINS gene expression were selected from 20 positive clones by r...