| Objectives With the purpose of seeking the effects of TSH and excessive KI on DUOX2 mRNA and H2O2,FRTL cells were intervened with high level of TSH and deceted with SYBR GREEN real-time PCR and Western Blotting.Methods 1?Effects of excessive KI on DUOX2 mRNA,Duox2 protein and H2O2 on FRTL cells:Treating FRTL cells with different concentration(10-5?5 ×10-5M?2.5 ×10-4M?1.25 × 10-3M)KI for 24h,changes of cell vitality were evaluated by methyl thiazolyl tetrazolium?MTT?colorimetry.After treatment for 24h,48h,FRTL cells were collected and got RNA.SYBR GREEN real-time PCR was used to analyse the level of DUOX2 mRNA;After treatment for 48h,72h FRTL cells were collected,fluorescence microscope was used to observed the expression of Duox2,western blotting was used to analyse the level of Duox2;Fluorospectro photometer was used to measure the quantity of H2O2 at 2h,24h.2.Effects of different concentrations of TSH on DUOX2 mRNA and H2O2 on FRTL cells:FRTL cells were cultured without TSH four days,and the concentration of FBS was maintained at 0.2%.Then treating FRTL cells with different concentration of TSH and KI(1 U/L TSH?10 U/L TSH?KI(10-3M)?KI+1 U/L TSH?KI+10 U/L TSH)for 24h,48h,the way of detection of DUOX2 mRNA and the quantity of H2O2 at 2h,24h,48h were the same with before.Results 1?Effects of excessive KI on DUOX2 mRNA,Duox2 protein and H2O2 on FRTL cells:?1?Effects of KI on vitality of FRTL cells:no detectable effects on cell vitality were found in 10-5M?2.5 ×10-4M KI groups?p>0.05?.1.25 ×10-3M KI groups was significantly decreased compared to control group?p<0.05?;?2?Effects of KI on expression of DUOX2 mRNA:After treatment different groups of KI for 24h,48h,RT-PCR showed that there was no significant difference in all groups?P>0.05?;?3?Effects of KI on expression of Duox2:After treatment different groups of KI for 48h,72h,Western Blotting showed that there was no significant difference in all groups?P>0.05?.No difference of Fluorescence intensity of Duox2 in cell membrane was detected with fluorospectro photometer;?4?Effects of KI on quantity of H2O2:After treatment different groups of KI for 2h,24h,there was no significant difference in the quantity of H2O2 of all groups?P>0.05?.2?Effects of different concentrations of TSH on DUOX2 mRNA and H2O2 on FRTL cells:?1?Effects of TSH on expression of DUOX2 mRNA:After treatment for 24h,the expression of 10U/L TSH group was higher than control group?P<0.05?,but 10-3mol/L KI group was lower than control group?P<0.05?;the expression of 1U/L TSH+10-3M KI group and 10U/L TSH+10-3M KI group were higher than KI group;After for 48h,the expression of 1U/L TSH group,10U/L TSH group,1U/L TSH+10-3M KI group,and 10U/L TSH+10-3M KI group were higher than control group?P<0.05?,the expression of 1U/L TSH+10-3M KI group and 10U/L TSH+10-3M KI group were higher than KIgroup,but 10-3 mol/L KI group was lower than control group?P<0.05?;?2?Effects of TSH on quantity of H2O2:After treatment for 2h,there was no effection on quantity of H2O2?P>0.05?;After treatment for 24h,the quantity of 10U/L TSH group washigher than control group,the expression of 1U/L TSH+10-3 M KI group and 10U/L TSH+10-3M KI group were higher than KI group?P<0.05?;10-3M KI group was lower than control group?P<0.05?;After treatment for 24h,the quantity of 1U/L TSH group and 10U/L TSH group were higher than control group?P<0.05?,the expression of 1U/L TSH +10-3,M KI group and 10 U/L TSH +10-3M KI group were higher than KI group;10-3M KI as lower than control group?P<0.05?.Conclusion1.When the level of TSH maintained at 1U/L,no effection were detected of KI with 10-5?1.25 × 10-3M on DUOX2 mRNA,Duox2 and H2O2;2.FRTL cells were cultured four days without TSH,we found KI can inhibit the expression of DUOX2 mRNA and production of H2O2,but TSH had a promoting action,and inhibitory action of KI can be weaken when FRTL cells were added with TSH,the combined effect showed promoting action. |
PDF Full Text Request