Expression of pEGFP-NeuroD recombinant plasmid in HepG2 cells and its function
AIM: To make gene tranafection with pEGFP-NeuroD expression plasmid containing enhanced green fluorescent protein, to observe its expression in HepG2 cells and to detect whether it could induce hepatocytes to secrete insulin in vitro. METHODS: 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 fluorescent protein expression was observed by fluorescence microscopy in the three groups and NeuroD-1 and insulin mRNA expression was detected by RT-PCR. Western Blot analysis was used to determine the expression of fusion protein NeuroD-EGFP. RESULTS: ① The recombinant plasmid pEGFP-NeuroD was successfully transfected into HepG2 cells. The strong expression of green fluorescent protein was observed by fluorescent microscopy and the transfection efficiency ranged from 30% to 40%. ② The expression of NeuroD-EGFP was detected by RT-PCR and Western blot in three groups. 634 bp NeuroD-1 DNA fragment was amplified by RT-PCR and the molecular weight of fusion protein NeuroD-EGFP was 67 × 103. But there was no significant difference in the protein expression among the three groups. ③ No evidence of insulin secretion of hepatoma carcinoma cells was shown by RT-PCR. CONCLUSION: 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 is not enough to induce hepatoma carcinoma cells to secrete insulin.
NeuroD-1Gene transfectionenhanced green fluorescent proteinHepG2 cellsinsulin
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维普
