Transfecting MIN6 cell line and enhancing cell viability by Reg3? cDNA / 西安交通大学学报(医学版)

ABSTRACT

Objective To create a MIN6 cell line overexpressing murine Reg3? cDNA and to investigate its cell viability character under low glucose concentrations.Methods The full-length murine Reg3? cDNA was inserted into the plasmid pcDNA3.1(-).Then MIN6 cells were transfected with the Reg3?-pcDNA3.1 and pcDNA3.1 vector alone to establish Reg3?-overexpression and empty vector MIN6 cell line.Reg3? protein in the low glucose concentration cell medium was detected by Western blot.Cell viability was evaluated by an MTT reduction conversion assay.Results Three Reg3?-overexpression MIN6 cells were confirmed by real-time PCR and Western blot.Reg3? expression was barely detectable in the cells transfected with the empty vector alone.In contrast,the levels of Reg3? mRNA and protein in three pcDNA-Reg3?-transfected clones were increased by an average 10-and 6-fold,respectively.Western blot also revealed Reg3? protein release into the culture medium.In MTT cell proliferation assay,compared to vector-transfection alone,three clones of Reg3?-overexpression MIN6 cells exhibited 2-fold increases in cell number over 7 days when cultured in the presence of 10 mL/L fetal bovine serum and 5.5 mmol/L glucose.Conclusion The Reg3?-overexpression MIN6 cells have been established.Reg3? protein was detectable in culture medium,supporting an endocrine action,and Reg3? overexpression promoted islet cell growth.