Regulation Of The Gene Expression Of Heme Oxygenase-1 And Its Potential Role In Treatment In Diabetic Vasculopathy

Objective To investigate the effect of high glucose on the expression of heme oxygenase-1(HO-1)in high glucose induced human umbilical vein endothelial cells(HUVECs),and observe the effect of HO-1 when it over-expressed to the function of HUVECs.Methods(1) Culture and identification of HUVECs in vitro.(2)The cells were treated with D-glucose at different concentrations(ranging from 5.6 to 45mmol/L)for 48h or exposed to 30mmol/L D-glucose for 0,24,48,72h,respectively.D-mannitol served as control.Gene expression was analyzed at mRNA level by RT-PCR or at protein level by Western blot. The trypan-blue exclusion test was used to study the influence of rosiglitazone on cell survial.(3)The cells cultured in high glucose concentration(30mmol/L)were treated with rosiglitazone at different concentrations(ranging from 5 to 30μmol/L)for 48h or exposed to 10μmol/L rosiglitazone for 0,24,48,72h,respectively.D-mannitol served as control.Gene expression was analyzed at mRNA level by Real time-PCR or at protein level by Western blot.The trypan-blue exclusion test was used to study the influence of rosiglitazone on cell survial.When rosiglitazone concentration rose,trypan-blue excluded cells were increased.(4)Construct and certificate eukaryotic expression plasmid carrying human HO-1 gene,transiently transfected the ECV304 cell line, and cell lysate was prepared for RT-PCR and Western blot,respectively. Results(1)Successfully cultured and certificated the HUVECs in vitro, the cells used in the experiment were all 3 to 5 generations.(2)Compared with normal group,the expression of HO-1 was inhibited by high D-glucose in a dose and time-dependent manner.When D-glucose concentration rose,trypan-blue excluded cells were decreased.(3) Rosiglitazone can induce the expression of HO-1 of HUVECs cultured in high glucose concentration in a dose and time-dependent manner.When rosiglitazone concentration rose,trypan-blue excluded cells were increased.(4)Successfully constructed and certificated the eukaryotic expression plasmid pCDNA3.1-HO-1.Conclusions High glucose could inhibit the expression of HO-1 gene in HUVECs in a dose and time-dependent manner,making the cell death of endothelial cells increase.Rosiglitazone could induce the expression of HO-1 gene in HUVECs cultured in high glucose condition.,making the cell death of endothelial cells decrease,which could protect the vascular function.