PAI-1反义RNA对人主动脉平滑肌细胞中血管内皮生长因子的影响

目的 探讨纤溶酶原激活物抑制剂－1（plasminogen activator inhibitor-1,PAI-1)反义RNA对离体培养的主动脉平滑肌细胞（smooth muscle cell,smc)PAI-1表达的作用及对血管内皮生长因子（vscular endothelial growth factor,VEGF)表达的影响。方法 PCR扩增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反应RNA重组质粒。将pcDNA3.1-反应PAI－1重组质粒转染SMC中。通过免疫组化、Western印迹、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含量最低，VEGF的表达也减少。第5天、PAI－1含量逐渐高，VEGF也相应增加。第7天、PAI－1含量接近于正常，VEGF也增至正常水平。结论 反义PAI－1RNA能有效阻断SMC中PAI－1的蛋白合成，同时抑制细胞中VEGF的表达。

Effect of PAI-1 antisense RNA on vascular endothelial growth factor expression in aorta smooth muscle cells cultured in vitro

OBJECTIVE: To investigate the role of plasminogen activator inhibitor-1(PAI-1) antisense RNA in regulating the expression of PAI-1 and vascular endothelial growth factor (VEGF) in aorta smooth muscle cells cultured in vitro. METHODS: The second extron of PAI-1 was amplified with polymerase chain reaction(PCR), the production was inserted into eukaryotic cell expression vector pcDNA3.1 after it had been purified and cloned so as to construct PAI-1 antisense RNA recombination plasmid. The recombination plasmid was transfected into SMC. PAI-1 expression was detected by immunohistochemistry, Western blod and ELISA; the effects of PAI-1 variation on VEGF was examined by immunofluorescence. RESULTS: PAI-1 antigen was the lowest in cells on the third day after transfection; the expression of VEGF was also decreased. PAI-1 antigen gradually increased on the fifth day and VEGF increased correspondingly. On the seventh day, PAI-1 antigen and VEGF increased to nearly normal level. CONCLUSION: PAI-1 antisense RNA can block the translation progress of PAI-1 proteins effectively and inhibit the expression of VEGF in aorta smooth muscle cells.