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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。
AIM: To investigate the role of the cis-regulatory element 5 ’upstream of EDN1 gene in EDN1 gene transcription in HUVEC induced by homocysteine (Hcy). Methods: The content of reactive oxygen species (ROS) in HUVECs was detected by fluorescent probe DCF. The expression of EDN1 mRNA was detected by RT-PCR. The expression of c-jun / AP-1 protein was detected by Western blotting. The secretion of EDN1 in the supernatant was measured. The AP-1 reporter gene activity of HUVECs was observed by transient transfection of recombinant plasmids. Results: Hcy could significantly increase the ROS production in HUVECs, promote the secretion of EDN1 and increase the expression of EDN1mRNA. Transient transfection assay showed that Hcy obviously induced the expression of pGL3-EDN1-AP-1 (-115 / +135) luciferase , But the basic expression of pGL3-EDN1-AP-1MU (-115 / +135) luciferase and the induction of Hcy of plasmid pGL3-EDN1-AP-1 (-115 / +135) reduce. CONCLUSION: It is hypothesized that Hcy may change the redox state in HUVECs, promote the release of ROS, activate the nuclear transcription factor AP-1 and regulate the gene transcription through AP-1 cis-acting element in EDN1 gene.