Expression Of PEGFP-NeuroD Recombinant Plasmid In HepG2 Cell And Its Function Study

ObjectiveThe helix-loop-helix (HLH) protein NEUROD-1 (also known as BETA2) ,functions as a transcription factor ,is essential for proper pancreatic islet morphogenesis and insulin gene transcription. It was demonstrated previously that the endocrine pancreas of BETA2/NeuroD-deficient mice undergoes massive apoptosis and, consequently, animals die of diabetes shortly after birth. Recently, Cao et al. demonstrated persistent expression of the pancreatic duodenal homeobox-1 (Pdx-1) transcription factor in hepatic cells, exposured to a hyperglycemia, reprograms these cells into pancreatic beta-cell precursors. This suggests that the ectopic insulin expression in the liver can be accomplished under certain conditions. In our study we make gene transfection with pEGFP-NeuroD expression plasmid containing enhanced green fluorescent protein,and then observe its expression in HepG2 cells and detect whether it could induce hepatocytes to secrete insulin in vitro.Methods1.The NeuroD-1 gene was amplified from recombinant plasmid pcr3.1-NeuroD template by PCR.Then it was inserted into pEGFP-C1 to form recombinant plasmid pEGFP-NeuroD ,which can express NeuroD-1 gene2. After identification, the recombinant plasmid pEGFP-NeuroD was transfected into HepG2 cells cultivated in three different concentrations of glucose culture solution by techniques of lipofectamine transfection. The green fluorescence protein expression was observed by fluorescence microscopy, and NeuroD-1 and insulin mRNA expression was detected by RT-PCR. At last Western blot analysis was used to determine the expression of fusion protein NeuroD-EGFP.Results1. The recombinant plasmid pEGFP-NeuroD was successfully constructed.2. The recombinant plasmid pEGFP-NeuroD was successfully transfected into HepG2 cells. The strong expression of green fluorescence protein was observed by fluorescence microscopy. The transfection efficiency ranged from 30% to 40%.The expression of NeuroD-EGFP was detected by RT-PCR and Western blot in three groups. About 634bp NeuroD-1 DNA fragment was amplified by RT-PCR and the molecular weight of fusion protein NeuroD-EGFP was 67KD.But there was no significant difference of the protein expression among the three groups.3. There was no evidence to indicate the insulin secretion of hepatoma carcinoma cells by RT-PCRConclusions1.To construct the fusion protein expression vector pEGFP-NeuroD making use of the characteristics of pEGFP-C1 with enhanced green fluorescent protein(EGFP)2.Recombinant plasmid pEGFP-NeuroD is effectively expressed after being transfected into HepG2 cells in vitro,and it suggests that the transfection only with NeuroD-1 expression plasmid may not be enough to induce hepatoma carcinoma cells to secrete insulin.