Effects Of XPD Gene On Apoptosis Of HUVEC Induced By Ox-LDL

OBJECTIVE:This study build the model of apoptosis of human umbilical vein endothelial cellby oxidized low density lipoprotein(ox-LDL), based on firstly discoverring that repairgene expression D (xeroderma pigmentosum D, XPD) was expressed in human veinendothelial cells (Human Umbilical Vein Endothelial Cell, HUVEC), to observe XPDexpression in vascular endothelial cells while undergoing apoptosis; at the same timesilencing XPD gene expression in endothelial cells, to survey ox-LDL-inducedapoptosis of endothelial cells, investigate the effect of XPD-siRNA on ox-LDL-induced apoptosis of endothelial cells and its mechanism.METHODS：1．Human Umbilical Vein Endothelial Cell were cultured, and XPD geneexpression was determined.2．Build the model of apoptosis of human umbilical vein endothelial cell byoxidized low density lipoprotein(ox-LDL), harvesting for24hours with finalconcentration of ox-LDL in the medium respectively at50μg/m l,100μg/m l,150μg/m l,200μg/m l. Cell viability was detected by MTT and the best concentration ofox-LDL was chosen to build the model of apoptosis of endothelial cell.3．The expression of XPD protein in HUVEC infulenced by ox-LDL wasdetected by western blotting.4．Three targeted siRNAs were designed and synthesized, they were siRNA-1,siRNA-2, siRNA-3, transfected into HUVEC with liposome. The expression of XPDprotein was detected by western blotting and the best one would be used for thesubsequent experiments.5．The right targeted siRNA was transfected into HUVEC, and then HUVECwas harvested for24hours with selected concentration of ox-LDL. Six groups weredivided: a. Blank Control Group, b. Negative Control siRNA Group, c. XPD-siRNAGroup, d. ox-LDL Group, e. ox-LDL+Negative Control siRNA Group, f. ox-LDL+ XPD-siRNAGroup.6．Cell viability and apoptosis rate were detected respectively by MTT andFlow Cytometry; protein of all groups was extracted to assessed XPD, Bcl-2, Baxby Western Blottig; mRNA of all groups was extracted to measured XPD, Bcl-2,Bax by Western Blottig.2. Statistical method: analyzed by SPSS13.0statistical software, experimentaldata were expressed as mean±SD. P <0.05was considered statistically significant.RESULTS:1. XPD was expressed in HUVEC.3. When HUVEC infulenced by ox-LDL in the concentration of50～200μg/mlfor24hours, the balance between activity and apoptosis, and the concentration of100μg/ml was the best one.4. The expression of XPD was increased in HUVEC after being inflenced bydifferent concentrations of ox-LDL.5. Three siRNAs can effectively inhibit the expression of XPD in HUVEC, andXPD-siRNA-2had the highest efficiency（76.15±2.14）%.6. The result of MTT show that, compared with blank control group, the vitalityof XPD-siRNA group was increased（140.43±4.26VS100, p<0.05）;compared withox-LDL+negative control siRNA group, the vitality of ox-LDL+XPD-siRNA wasalso increased（110.91±3.22VS70.58±4.67, p<0.05）.7. The outcome of flow Cytometry displayed that, compared with blank controlgroup, the apoptosis rate of XPD-siRNA group was reduced（2.0±0.30VS5.17±0.16,p<0.05）;compared with ox-LDL+negative control siRNA group, the vitality ofox-LDL+XPD-siRNA was absolutely reduced（15.26±0.74VS28.32±0.63, p<0.05）.8. The consequence of RT-PCR and Western Blotting indicated that, comparedwith blank control group, the expression of XPD in XPD-siRNA group wasdeclined((0.11±0.01VS0.32±0.01, p<0.05）and（0.1±0.0.01VS0.49±0.0.01, p<0.05));the expression of Bcl-2was elevated((0.35±0.02VS0.15±0.02, p<0.05） and（0.24±0.0.02VS0.07±0.02, p<0.05)); the expression of Bax was fell((0.1±0.01VS0.53±0.02, p<0.05）and（0.07±0.01VS0.37±0.02, p<0.05)).The consequence of RT-PCR and Western Blotting also manifest that, compared with ox-LDL+negative control siRNA group, the expression of XPD in ox-LDL+XPD-siRNA group was dropped(（0.45±0.02VS0.65±0.01, p<0.05） and（0.25±0.0.01VS1.06±0.01, p<0.05）; the expression of Bcl-2wasincreased（(0.24±0.02VS0.09±0.06, p<0.05）and (0.15±0.01VS0.04±0.00, p<0.05）);the expression of Bax was decrease(0.49±0.03VS0.89±0.01, p<0.05）and （0.19±0.01VS0.83±0.01, p<0.05）).CONCLUSION:1. XPD was expressed in HUVEC, and its expression was upgraded afterHUVEC being infulenced by ox-LDL.2. XPD participated in the apoptosis of HUVEC induced by ox-LDL.3. The expression of Bax and Bcl-2may be associated with the possiblemechanism of XPD participating in the apoptosis of HUVEC induced by ox-LDL.