中文摘要：

目的：研究E1A激活基因阻遏子蛋白（CREG）在依托泊苷（VP-16）诱导的人脐静脉内皮细胞（HUVECs）凋亡中的作用及其调控机制。方法：将体外培养的原代HUVECs用pLNCX-CREG和pLXSN-shRNA-CREG反转录病毒真核表达载体分别转染,通过G418（800 mg/L）和嘌呤霉素（2.5 mg/L）筛选分别获得CREG过表达组HUVECs（H-C）和CREG低表达组HUVECs（H-S）。在H-C、HUVECs（H）和H-S3种细胞模型中加入凋亡诱导剂VP-16（100μmol/L,6 h）,用TUNEL法和流式细胞术检测细胞的凋亡情况;用Western blotting检测细胞中磷酸化p38 MAPK（p-p38 MAPK）的蛋白表达量;应用p38特异性抑制剂SB203580（20μmol/L）预处理H-S细胞后,检测VP-16刺激诱导的细胞凋亡情况。结果：HUVECs经pLNCX-CREG和pLXSN-shRNA-CREG反转录病毒真核表达载体转染获得稳定的细胞克隆。经Western blotting证实,与HUVECs对照比较,H-C组细胞CREG表达明显增加至160%,而H-S组细胞中的CREG表达下降约70%。加入凋亡诱导剂VP-16（100μmol/L,6 h）后,TUNEL和流式双荧光染色检测结果均提示H-S中的细胞凋亡率明显升高,而H-C组细胞凋亡率较HUVECs对照组明显降低,两者具有显著差异。进一步应用Western blotting分析显示经VP-16刺激后,H-S细胞中p-p38蛋白表达较HUVECs和H-C组细胞显著增加;在H-S中添加SB203580（20μmol/L）后,VP-16诱导的细胞凋亡现象被显著抑制。结论：CREG过表达可能通过抑制p38 MAPK的激活阻遏VP-16诱导的HU-VECs凋亡。

英文摘要：

AIM： To study the effect of cellular repressor of E1A-stimulated genes（CREG） and its mechanism on apoptosis of human umbilical vein endothelial cells（HUVECs） induced by etoposide（VP-16）.METHODS： Primary HUVECs were cultured.RetroviraI eukaryotic expression vectors pLNCX-CREG and pLXSN-shRNA-CREG were transfected into HUVECs.The stable cell clone was selected and obtained by screening with G418（800 mg/L） and the puromycin（2.5 mg/L）,respectively.CREG expression was detected by Western blotting.The cells with overexpression of CREG（H-C） and those with CREG down-regulation（H-S） were pretreated with apoptotic inducer VP-16 at 100 μmol/L for 6 h.The apoptotic rates of the 3 kinds of cells were analyzed by TUNEL and flow cytometry with annexin V/PI dualstaining.Furthermore,the protein levels of phosphorylated p38 mitogen-activated protein kinase（p-p38 MAPK） in the 3 kinds of cells were analyzed by Western blotting.The p38-specific inhibitor SB203580（20 μmol/L）was used to investigate the effects of p-p38 expression on apoptosis.RESULTS： Western blotting showed that CREG expression was obviously increased up to 160% in H-C compared to HUVECs.However,CREG expression in H-S cells was identified to be down-regulated to 70% compared with HUVECs.TUNEL assay and annexin V/PI dual-color FACS showed that the apoptotic rate was dramatically increased in H-S cells,but decreased in H-C cells.Subsequently,Western blotting exhibited that p-p38 expression was increased in H-S cells compared to HUVECs and H-C cells.When the H-S was pretreated with SB203580,the apoptotic rate was decreased.CONCLUSION： CREG overexpression might prevent HUVECs from apoptosis by inhibiting p38 MAPK activition.