ABSTRACT
BackgroundDiabetic retinopathy (DR) associated with this disease closely approximates the oxidative damage inflicted on retinal pigment epithelial (RPE) cells by high glucose,in recent years,people devote themselves to the protection of RPE cells extensively.ObjectiveTo investigate the protective effect of pancreatic β MIN-6 cells on retinal pigment epithelial(RPE) cells from high glucose-induced damage.MethodsRPE cells were incubated with normal medium for 4 d and divided into 3 groups:normal glucose group,high glucose group and MIN-6 cells group.RPE cell were exposed with 5 mmol/L normal glucose in normal glucose group,exposed with 30 mmol/L high glucose in high glucose group,and exposed with 5 × 104 MIN-6 cells and 30 mmol/L high glucose in MIN-6 cells group.After 24 h,cell viability of RPE cells was determined by MTT cell viability assay.Results More than 95％ cells showed the brown staining by ABC method.After incubation for another 24 hours,the A550 value was 0.44±0.02,0.30±0.01 and 0.41±0.01 in the normal glucose group,high glucose group and MIN-6 cells group respectively with a significant difference among these three groups (F =19.94,P＜ 0.01 ).The A55o value was significantly higher in the normal glucose group and the MIN-6 cells group compared with the high glaucose group (t =6.85,5.62,P＜0.01 ).The survival rate of RPE cells in the normal glucose group was(97.5±3.3 )％,and that in the high glucose group was ( 68.2 ± 4.5 ) ％,showing significant difference between them ( t =11.30,P＜0.01 ).ConclusionsHigh glucose-induced damage of RPE cells is abrogated,and MIN-6 cells can protect RPE cells from high glucose-induced damage.
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.