Role Of Different Concentration Of D-glucose And Insulin On Expression Of TGF-β₁ In HK-2 Cells

Objective: To examine whether D-glucose and insulin can affect the expression of TGF- β₁ in HK-2 cells and the interaction between them.Methods:All experiments were performed using HK-2 cells under serum free conditions. According to the concentration of D-golucose and insulin in culture medium, growth-arrested cells were devided into 6 groups. A:(control): DMEM/F-12+5mmol/L D-golucose+ 5uIU/mL insulin. B: DMEM/F-12+5mmol/L D-golucose+ 125uIU/mL insulin. C: DMEM/F-12+25mmol/L D-golucose +125uIU/mL insulin. D: DMEM/F-12+25mmol/L D-golucose+1.25uIU/mL insulin. E:DMEM/F-12+25mmol/L D-golucose.F:DMEM/F-12+125uIU/mL insulin. After added to different culture medium, cells were cultured 12h,24h,48h,72h ,supernatant were collected through the subsequent 72 hours and stored at -70℃ befor detected. In all experiments TGF- β₁ protein concentration in the supernatant was determined by specific enzyme-linked immunosorbent assay(ELISA).Subsequently,cells were counted and total RNA was isolated and then TGF-β₁ mRNA and GAPDH mRNA examined by reverse transcriptase-polymerase chain reaction (RT-PCR).Both TGF- β₁ and GAPDH PCR product were separated by flat bed electrophoresis in 2% agrose gels,stained with ethidium bromide(EB) and photographed. The nagative were scanned using a densitometer and the density of the bands compared to those of the housekeeping gene GAPDH. The data are presented as means ±SD of n experiments. Statistical analysis was performed using the unpaired Student' s t-test and One-Way ANOV-A, with a value of P<0.05 considered to represent a significant differernce.Results: All samples were detected TGF- β₁ mRNA and TGF- β₁ protein. (1) In C and D groups(both include 25mmol/L D-glucose in culture medium) mRNA expression levels increased since 12h affer addition experimental medium, and reached the peak at 24h and kept the peak in subsequent 48h. After addition experimental medium 24h ,C, D groups TGF- β₁ mRNA levels were higher significantly than A group(0.8272± 0.0325; 0.8276±0.0239 vs 0.5288±0.0175 F=276.9; P=0. 000)while there was no difference between C and D groups.However between B and A groups in the 72h ,there was no difference in TGF-β₁ mRNA levels (Afer exposure 24h, 0.5438 ±0.0083 vs 0.5288 ± 0.0175 t =1.267 P=0. 086).(2) In B and C groups(125uIU/mL insulin in both medium),...