E1A激活基因细胞阻遏子蛋白在人脐静脉内皮细胞凋亡中的作用  

Effect of cellular repressor protein of E1A-stimulated genes on the apoptosis of human umbilical vein endothelial cells

作　　者：陶杰[1] 闫承慧[2] 郭鹏[2] 吴光哲[2] 王占胜[2] 韩雅玲[2] 

机构地区：[1]解放军第四军医大学西京医院心内科,陕西省西安市710032 [2]解放军沈阳军区总医院心内科,辽宁省沈阳市110016

出　　处：《中国组织工程研究与临床康复》2009年第37期7281-7285,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金面上项目(30770793);国家杰出青年科学基金项目(30800465)~~

摘　　要：背景:研究内皮细胞凋亡的机制及其与动脉硬化形成的关系将为动脉粥样硬化的防治提供新的思路。而E1A激活基因的细胞阻遏子蛋白在内皮细胞凋亡中的作用,目前尚未见报道。目的:探讨E1A激活基因的细胞阻遏子蛋白在去血清诱导的人脐静脉内皮细胞凋亡中的作用及其调控机制。时间及地点:实验于2007-10/2008-12在解放军沈阳军区总医院全军心血管病研究所实验室完成。材料:人脐静脉内皮细胞,Phoenixamphotropic293细胞,pLNCX-CREG和pLNCX-GFP反转录病毒真核表达载体。方法:采用持续去血清培养的方法诱导内皮细胞凋亡,用TUNEL染色和Westernblot检测去血清饥饿24,48,72h后各实验组细胞中cleave-caspase3表达;用pLNCX-CREG和pLNCX-GFP反转录病毒真核表达载体转染人脐静脉内皮细胞,并筛选稳定表达的细胞克隆;应用Westernblot检测E1A激活基因的细胞阻遏子蛋白的表达,并进一步应用流式双荧光染色分析E1A激活基因的细胞阻遏子蛋白过表达在去血清诱导的人脐静脉内皮细胞凋亡中的作用。主要观察指标:cleave-caspase3表达量,人脐静脉内皮细胞凋亡率。结果:成功构建了E1A激活基因细胞阻遏子过表达的人脐静脉内皮细胞模型,证实内皮细胞E1A激活基因细胞阻遏子的表达随凋亡的增加而增加,E1A激活基因细胞阻遏子过表达的人脐静脉内皮细胞去血清饥饿后凋亡较对照组明显减少。结论:E1A激活基因细胞阻遏子过表达抑制了去血清诱导的人脐静脉内皮细胞凋亡,机制可能与调控PI3K蛋白表达有关。BACKGROUND： Researches on the mechanism of vascular endothelial cells’ apoptosis and its relation with atherosclerosis will provide new ideas for the prevention and cure of atherosclerosis. Cellular repressor of E1A-stimulated genes （CREG） protein’s effect on vascular endothelial cells’ apoptosis is unknown now. OBJECTIVE： To explore the CREG protein’s effects and its mechanism on regulating the apoptosis of human umbilical vein endothelial cells （HUVEC） induced by cultivation with 1% serum medium. DESIGN, TIME AND SETTING： The experiment was performed in the laboratory of Cardiovascular Institute of Chinese PLA, General Hospital of Shenyang Military Area Command of Chinese PLA from October 2007 to December 2008. MATERIALS： HUVEC, Phoenix amphotropic 293 cells, pLNCX-CREG and pLNCX-GFP retroviral eukaryotic expression vectors. METHODS： The HUVEC were starved by exposure to 1% serum medium for 24, 48, 72 hours, and apoptosis was analyzed by TUNEL staining and Western blot which detected the expression of cleave-caspase3. Retrovirus eukaryotic expression vectors pLNCX-CREG and pLNCX-GFP were transfected to HUVEC, and cell clones that expressed stably were selected. The expression of CREG was analyzed by Western blot, the effect of over-expression of CREG on apoptosis of HUVEC that induced by starved cultivation with 1% serum medium was further analyzed by flow cytometry after fluorescent staining. MAIN OUTCOME MEASURES： Expression of cleave-caspase3, apoptosis ratio of HUVEC. RESULTS： The CREG over-expressed HUVEC model was constructed successfully, demonstrating CREG expression increased with the augmentation of apoptosis and apoptosis of over-expressing CREG HUVEC after starved cultivation with 1% serum medium decreased compared with control group. CONCLUSION： CREG overexpression inhibits the apoptosis of HUVEC, and the mechanism is possibly concerned with its regulation on the expression of phosphoinositide 3-kinase protein.

关 键 词：E1A激活基因细胞阻遏子 凋亡 人脐静脉内皮细胞 PI3K 

分 类 号：R318[医药卫生—生物医学工程]