| Objective We cloned the fragment of SelS gene from human adipose tissue and constructed the pcDNA3.1-SelS eukaryotic expression vector. After pcDNA3.1-SelS and pcDNA3.1 were transient transfected into ECV304 cells, which were divided into three groups, the expression of SelS mRNA and protein were determined respectively. The oxidative markers were assayed to investigate the inhibitive effect of SelS gene to H2O2-mediating injuring in three groups of ECV304 cells. We assayed the mRNA expression before and after damage induced by H2O2 in three groups of ECV304 cells to discuss whether the protection effect of SelS gene is associated with Caveolin-1 and give a further possible mechanism of SelS gene protecting ECV304 cells from oxidative stress.Methods1. SelS gene segment from human adipose tissue was amplified by RT-PCR. Firstly, SelS gene segment was cloned into pMD18-T vector to construct a cloned vector. Secondly, pMD18-SelS was incubated in presence of EcoRI/XhoI and then ligated into eukaryotic expression vector pcDNA3.1. The plasmid was named as pcDNA3.1-SelS and finally identified by EcoRI/XhoI digestion and sequence.2. The plasmid pcDNA3.1-SelS and pcDNA3.1 were both transient transfected into ECV304 cells by using the lipofectamineTM 2000. ECV304 cells were divided into transfected group (transfected with pcDNA3.1-SelS), empty vector group (transfected with pcDNA3.1) and non-transfected group. Then we verified SelS gene expression in ECV304 cells by RT-PCR and Western blot. 3. After the transient transfection for 24 hours in 24-well plate, three groups cells were cultured by fresh serum-free medium IMEM containing 0,400,600,800,1000μmol/L H2O2 for 6 hours. The supernatant of culture medium were collected, then cell viability was measured by MTT assay, the intracellular maleic dialdehyde (MDA) was detected by TBA method and the antioxidant ability of superoxide dismutase (SOD) was assayed by xanthine oxidase technique.4. Three groups of cells were cultured by fresh serum-free medium IMEM containing 800μmol/L H2O2 for 6 hours. The expression of Caveolin-1 mRNA in three groups was determined by RT-PCR.Results1. The electrophoresis results showed the extraction of total RNA from human adipose tissue was complete. The EcoRI/XhoI digestion and sequence results showed that SelS gene segment had been successfully cloned into eukaryotic expression vector pcDNA3.1.2. After the transfection, RT-PCR and Western blot results showed that endothelial cells have endogenous SelS gene expression and which expression was obviously increased in transfected group as compared with that in non-transfected group and empty vector group (P<0.01). There was no obvious difference between non-transfected group and empty vector group (P>0.05).3. Three groups of cells were cultured by fresh serum-free medium IMEM containing 0, 400, 600, 800, 1000μmol/L H2O2 for 6 hours. The cell survival rates were decreased. At 800 and 1000μmol/L H2O2 concentration, cell survival rates in transfected group (34±10, 27±15) were much higher than non-transfected group (32±12, 24±9) and empty vector group (31±14, 23±10) (P<0.01). There was no obvious difference between non-transfected group and empty vector group (P>0.05).4. MDA content in the culture media of three group of cells were increased in a H2O2 concentration-dependent manner. At 600, 800 and 1000μmol/L H2O2 concentration, MDA content (nmol/mL) in transfected group (3.50±0.07, 6.30±0.07, 10.50±0.08) were lower than non-transfected group (6.40±0.03, 10.40±0.11, 18.00±0.07) and empty vector group (5.80±0.08, 9.00±0.09, 16.40±0.19) (P<0.05, P<0.05, P<0.01). The differences became obvious at 1000μmol/L H2O2 concentration (P<0.01). There was no obvious difference between non-transfected group and empty vector group (P>0.05).5. SOD activity in the culture media of decreased with the increasement of H2O2 concentration. At 600, 800 and 1000μmol/L H2O2 concentration, SOD activity (U/ml) in transfected group (8.34±0.16, 7.48±0.87, 6.60±1.58) were lower than that in non-transfected group (7.52±0.29, 5.70±0.22, 4.62±2.33) and empty vector group (7.12±1.63, 6.10±1.78, 5.00±1.27) (P<0.05, P<0.01, P<0.01). The differences became obvious at 800 and 1000μmol/L H2O2 concentration (P<0.01). There was no obvious difference between non-transfected group and empty vector group (P>0.05).6. RT-PCR assayed the expression of Caveolin-1 mRNA before and after H2O2 treatment respectively. Three groups cells were cultured by fresh serum-free medium IMEM containing 0, 400, 600, 800, 1000μmol/L H2O2 for 6 hours, Caveolin-1 mRNA expression were all increased in non-transfected group (0.74±0.01 vs 0.48±0.01, P<0.01), empty vector group (7.12±1.63 vs 0.58±0.01,P<0.05) and transfected group (0.67±0.01 vs 0.63±0.01,P<0.01),compared with that before H2O2 treatment (P<0.01,P<0.05 , P<0.01). After treated with H2O2, Caveolin-1 expression was increased dramatically, Caveolin-1 mRNA expression in transfected group (0.67±0.01) were lower than that in non-transfected group (0.74±0.01) ( P<0.01) and empty vector group (7.12±1.63) (P<0.01). There was no obvious difference between non-transfected group and empty vector group (P>0.05).ConclusionsWe successfully constructed the pcDNA3.1-SelS eukaryotic expression vector by transient transfection into ECV304. It is confirmed that endothelial cells have endogenous SelS gene expression. Elevated SelS gene expression can attenuate H2O2 inducing inhibitive effect of proliferative activity in ECV304 cells, reduce the production of MDA, and enhance the activity of SOD. H2O2 can up-regulate Caveolin-1 expression in ECV304 cells, while overexpressing SelS gene may depress this elevation. It is demonstrated that SelS gene play a positive role in endothelial protection and might have some relationship with Caveolin-1. |
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