The Pro-Apoptosis Effects Of Xeroderma Pigmentosum D Gene On Human Umbilical Vein Endothelia Cells

OBJECTIVE:To observe whether xeroderma pigmentosum D(XPD) gene is expressed inhuman umbilical vein endothelia cells (HUVECs); to investigate pro-apoptosis effectsof xeroderma pigmentosum D(XPD) gene on human umbilical vein endothelia cells(HUVECs).METHODS:1.Human Umbilical Vein Endothelial Cell (HUVEC) were cultured,and after48-72hours cells mRNAs were extracted.XPD gene expression was determined byRT-PCR.2. Recombinant plasmid pEGFP-N2/XPD transfect HUVEC. Shaked bcteria andextracted plasmids:recombinant plasmid pEGFP-N2/XPD and vacant vector plasmidpEGFP-N2.The two plasmids were transient transfected into HUVEC by liposome2000method,with the same genetic background and algebra HUVEC as blankcontrols.After48hours,the expression of green fluorescent protein was observedthrough fluorescence microscopy. Through RT-PCR and Western blot, the expressionlevels of XPD further confirmed that whether the transfection was successful.3. The experiments were divided into three groups:(1)control group;(2)pEGFP-N2group;(3)pEGFP-N2/XPD group. The pro-apoptosis effects of XPD Geneon Human Umbilical Vein Endothelia Cells were detected through these methods,.(1).Cells were transient transfected in96-well plates,and the proliferationactivity of cells in each group was detected by MTT.(2).After cells were transient transfected,the cell apoptosis rate of each groupwas examined by flow cytometry.(3).Cells mRNAs were extracted.Through RT-PCR the mRAN expression levelsof XPD,Bcl-2,Bax and wt-p53were detected.(4).Cells proteins were extracted.Through Western blot the protein expressionlevels of XPD,Bcl-2,Bax and wt-p53were detected.RESULTS: 1. There was XPD expression in the cultured HUVEC.2. Green fluorescences were observed in the cells transfected with pEGFP-N2/XPD or pEGFP-N2;RT-PCR results and Western blot results showed that thetransfection of pEGFP-N2/XPD upregulated the expression of XPD compared tocontrol group and pEGFP-N2group (1.104±0.098VS0.273±0.029or0.295±0.031,0.861±0.068VS0.351±0.029or0.373±0.031,P<0.05);there was no statisticalsignificance between control group and pEGFP-N2group（P>0.05）.Though resultsindicating that the plasmids were transfected successfully.3. MTT results showed that the transfection of pEGFP-N2/XPD inhibited thecell growth compared to control group and pEGFP-N2group (52.35%VS100%or95.89%,P<0.05); there was no statistical significance between control group andpEGFP-N2group（P>0.05）.4. Flow cytometry results showed that the transfection of pEGFP-N2/XPDincreased the apoptosic rate of HUVEC compared to control group and pEGFP-N2group (16.43±3.57%VS2.41±1.01%or3.76%±1.27,P<0.01); there was no statisticalsignificance between control group and pEGFP-N2group（P>0.05）.5.RT-PCR and Western blot results showed that the transfection of pEGFP-N2/XPD decreased the mRNA expression of Bcl-2, increased the expression of Baxand wt-p53compared to control group and pEGFP-N2group(0.017±0.004VS0.140±0.016or0.121±0.014,0.911±0.126VS0.492±0.051or0.520±0.055,0.993±0.122VS0.352±0.037or0.371±0.041, P<0.05or P<0.01); the same as the proteinresults.There was no statistical significance between control group and pEGFP-N2group（P>0.05）.CONCLUSION:1. For the first time, we find that there is XPD expression in HUVEC.2. Upregulating the expression of XPD can promote HUVEC apoptosis.3. XPD may through regulate the apoptotic gene expression of Bcl-2, Bax andwt-p53to prompte HUVEC apoptosis.