【摘要】 目的 :探讨弱氧化修饰低密度脂蛋白 (mmLDL)对人脐静脉内皮细胞 (HUVEC)PAI- 1活性和mRNA表达的影响及其转录调控机制。方法 :人脐静脉内皮细胞的培养和鉴定。用发色底物法测定PAI- 1活性。North ern印迹分析法检测PAI- 1mRNA的水平。采用基因重组技术构建含不同长度PAI- 15’上游序列的荧光素酶报告基因质粒 ,瞬时转染进入内皮细胞 ,并检测荧光素酶的表达情况。利用PCR和测序技术 ,对构建质粒上AP - 1元件进行定点突变。Westernblot印迹杂交检测内皮细胞核内激活蛋白 - 1(AP - 1)蛋白水平。结果 :5 0mg/LmmLDL诱导HUVECsPAI - 1活性和mRNA表达量明显增高 ,同时提高核内AP - 1蛋白水平。mmLDL显著诱导构建质粒pGL3-PAI- 1- 15 0 9/+90和pGL3-PAI- 1- 82 3/+90的荧光素酶活性 ,但对质粒pGL3-PAI - 1- 5 5 3/+90和pGL3-PAI- 1- 4 7/+90诱导作用不明显。当PAI- 15’上游序列的 3个AP - 1元件突变后 ,mmLDL的诱导作用明显降低。结论 :(1)mmLDL增强血管内皮细胞PAI- 1活性与mRNA表达 ;(2 )PAI- 1活性提高与其mRNA表达增加呈正相关 ;(3)PAI- 15’上游序列中 3个AP - 1元件在mmLDL对PAI- 1诱导中具有重要调控作用。更多还原

【Abstract】 AIM: To study the influence of minimal modified low density lipoprotein (mmLDL) on plasminogen activator inhibitor-1 (PAI-1) activity, gene expression and regulation in human vascular endothelial cells. METHODS: The PAI-1 activity in human umbilical vein endothelial cells (HUVEC) culture medium was measured by chromogenic assay. The PAI-1 mRNA expression were determined by Northern blot. Using gene recombination techniques, four luciferase reporter gene plasmids containing different length of human PAI-1 gene promoter were constructed. Through the transient transfection analysis, the roles of AP-1 element(from -823 bp to -553 bp) in PAI-1 promoter have been determined. In order to further verify the role of AP-1 element, the three site-directed mutants were received using PCR and sequencing assay. RESULTS: The PAI-1 activity and mRNA level were increased when HUVECs were exposed to 50 mg/L mmLDL. At the same time, the AP-1 protein level was increased in nuclear. The induction by mmLDL were decreased markedly when the three AP-1 elements in PAI-1 promoter had been mutated, respectively. CONCLUSION: (1) mmLDL increased PAI-1 activity and mRNA expression in HUVEC. (2) Increase in PAI-1 activity induced by mmLDL was related to its mRNA expression. (3) Three AP-1 element in PAI-1 promoter may have an important role in PAI-1 gene transcription in endothelial cells induced by mmLDL.更多还原