【摘要】 目的:探讨EDN1基因5′上游AP-1顺式调控元件在同型半胱氨酸(Hcy)诱导HUVECs EDN1基因转录中的作用。方法:用荧光探针DCF检测HUVECs内活性氧的含量;用RT-PCR法检测EDN1mRNA的表达,用Western blotting法测定c-jun/AP-1蛋白的表达;同时用双抗夹心法测定HUVECs上清液中EDN1的分泌;利用重组质粒的瞬时转染技术观察了HUVECs的AP-1报告基因的活性。结果:Hcy能明显增加HUVECs ROS的生成,促进EDN1的分泌和提高EDN1mRNA的表达;瞬时转染分析实验结果显示Hcy能明显诱导质粒pGL3-EDN1-AP-1(-115/+135)荧光素酶的表达,但对质粒pGL3-EDN1-AP-1(-115/+135)的突变体pGL3-EDN1-AP-1MU(-115/+135)荧光素酶的基础表达及Hcy的诱导作用均明显降低。结论:推测Hcy可能改变HUVECs内的氧化还原状态,促进ROS的释放,激活核转录因子AP-1,通过EDN1基因中AP-1顺式作用元件调控该基因转录。更多还原

【Abstract】 AIM:The objective of this study was to observe the effects of AP-1 element on endothelin-1(EDN1)gene transcription in homocysteine(Hcy)-induced human umbilical vein endothelial cells(HUVECs).METHODS:The redox level in Hcy-induced HUVECs was determined by using the fluorescent product DCF.The influences of Hcy on expressions of EDN1 mRNA and protein of c-jun/AP-1 in HUVECs were measured by the methods of RT-PCR and Western blotting,respectively.The EDN1 secretion level of HUVECs induced by Hcy was determined by enzymatic immunoassay.The transient transfection assay was performed and luciferase activity of transcriptional factor AP-1 reporter gene induced by Hcy was detected.RESULTS:The Hcy significantly increased EDN1 secretion,EDN1 mRNA expression and oxidative stress in HUVECs.Hcy remarkably induced the expression of pGL3-EDN1-AP-1(115/+135)luciferase reporter gene plasmid.However,basic expression of mutation of pGL3-EDN1-AP-1(-115/+135)luciferase reporter gene plasmid was downregulated markedly in HUVECs induced by Hcy.CONCLUSION:Redox-active and release of ROS are changed by Hcy.Activated AP-1 element plays an important role in EDN1gene transcription in HUVECs induced by Hcy.更多还原