纤溶酶原激活物抑制剂-1反义RNA对主动脉内皮细胞部分功能的影响

目的 探讨纤溶酶原激活物抑制剂－1（plasminogen activator inhibior-1,PAI-1）反义RNA对离体培养的主动脉内皮细胞（endothelial cell,EC）PAI－1表达的作用及对血管内皮生长因子（vascular endothelial growth VEGF）的影响。方法 聚合酶链反应（PCR）手增PAI－1第2外显子，将PCR产物纯化克隆后连入真核细胞表达载体pcDNA3．1（－），构建PAI－1反义RNA重组质粒。将pcDNA3．1－反义PAI－1重组质粒转染EC中。通过免疫组化、免疫印迹法（Westernblot）、双抗体夹心法（ELISA）检测细胞中PAI－1表达的改变，通过免疫荧光技术观察细胞中PAI－1表达量的变化对VEGF的影响。结果 转染后第3天，细胞中PAI－1含量为0．017ng/ml，细胞浆内代表VEGF的绿色荧光最弱，表明VEGF的表达也减少；第5天，PAI－1含量为0．093ng/ml，细胞浆内绿色荧光物质略增加。第7天，PAI－1含量为0．143ng/ml，接近正常值，VEGF的荧光染色接近于正常水平。结论 反义PAI－1RNA能有效阻断EC中PAI－1的蛋白合成，同时抑制剂细胞中VEGF的表达。

Effects of plasminogen activator inhibitor-1 antisense RNA on aorta endothelial cells cultured

Objective To investigate the role of antisense RNA of plasminogen activator inhibitor 1 (PAI 1) in regulating the expression of PAI 1 and vascular endothelial growth factor (VEGF) in aorta endothelial cells (EC) cultured in vitro. Methods The second extron of PAI 1 was amplified by polymerase chain reaction(PCR) and the product was inserted into eukaryotic cell expression vector pcDNA3.1(-) to construct PAI 1 antisence RNA recombinant plasmid. The recombinant plasmid was transfected into EC and the PAI 1 expression was detected by immunohistochemistry, Westernblot and ELISA. The effects of PAI 1 variation on VEGF were examined by immunofluorescence method. Results PAI 1 antigen was the lowest (0 017 ng/ml) in cells and the immunofluorescence representing the expression of VEGF in the cytoplasm showed the weakest at the third day after transfection. At the fifth day, PAI 1 antigen increased to 0 093 ng/ml with VEGF expression increased correspondingly. At the seventh day, PAI 1 antigen(0 143 ng/ml) and VEGF increased closed to normal level. Conclusions PAI 1 antisense RNA blocked the translation of PAI 1 proteins effectively and inhibited the expression of VEGF in aorta endothelial cells.