【摘要】 探讨肿瘤坏死因子α对人脐静脉内皮细胞纤溶酶原激活物抑制剂 1活性和mRNA表达的影响 ,以及纤溶酶原激活物抑制剂 1基因 5’上游序列的调控元件在该基因转录调控中的作用。体外培养人脐静脉内皮细胞 ,加入不同浓度肿瘤坏死因子α作用不同时间。采用发色底物法测定纤溶酶原激活物抑制剂 1活性 ,Northern印迹分析法测定纤溶酶原激活物抑制剂 1mRNA水平。基因重组技术构建四个含人纤溶酶原激活物抑制剂 1基因不同长度5’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 ,并测定荧光素酶的表达情况。运用聚合酶链反应和序列分析法对构建质粒上的三个AP 1元件分别进行定点突变。结果发现 ,不同浓度肿瘤坏死因子α作用人脐静脉内皮细胞后 ,纤溶酶原激活物抑制剂 1活性和mRNA表达量均明显增高 ;当转染质粒含有纤溶酶原激活物抑制剂 1基因上游序列 - 15 0 9 +90和 - 82 3 +90时 ,肿瘤坏死因子α使转染细胞的荧光素酶表达量比对照组明显增高 ;当转染质粒含有 - 5 5 3 +90和 - 4 7 +90时 ,肿瘤坏死因子α的诱导作用不明显。在纤溶酶原激活物抑制剂 15’上游序列的三个AP 1元件突变后 ,肿瘤坏死因子α的诱导作用明显降低。结果提示 ,肿瘤坏死因子α增强血管内皮细胞纤溶酶原激活物抑制剂 1活性与mRNA表达 ;?更多还原

【Abstract】 Aim To study the influence of tumor necrosis factor α (TNF α) on plasminogen activator inhibitor 1 (PAI 1) activity, gene expression and regulation in human umbilical vein endothelial cell (hUVEC). Methods The PAI 1 activity in 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 polymerase chain reaction (PCR) and sequencing assay. Results The PAI 1 activity and mRNA level increased when hUVEC were exposed to 100 ku/L TNF α. At the same time, the AP 1 protein level increased in nuclear. The induction by TNF α decreased markedly when the three AP 1 elements in PAI 1 promoter have been mutated respectively. Conclusions TNF α could induce PAI 1 activity and mRNA in hUVEC; Increase of PAI 1 activity induced by TNF α was related to its mRNA expression; Three AP 1 elements in PAI 1 promoter may have an important role in PAI 1 gene transcriptions in endothelial cells induced by TNF α.更多还原