Fu L, Mei Y, Li H
Department of Cardiology of the First Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang 150001 P. R. China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2001 Apr;18(2):110-3.
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.
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.
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.
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.
探讨纤溶酶原激活物抑制剂-1（PAI-1）反义RNA在体外培养的主动脉平滑肌细胞中对PAI-1及血管内皮生长因子（VEGF）表达的调控作用。
采用聚合酶链反应（PCR）扩增PAI-1第二外显子，产物经纯化克隆后插入真核细胞表达载体pcDNA3.1，构建PAI-1反义RNA重组质粒。将重组质粒转染至平滑肌细胞（SMC）。采用免疫组织化学、蛋白质印迹法（Western blot）及酶联免疫吸附测定（ELISA）检测PAI-1表达；通过免疫荧光检测PAI-1变化对VEGF的影响。
转染后第3天细胞中PAI-1抗原最低；VEGF表达也降低。第5天PAI-1抗原逐渐增加，VEGF相应增加。第7天，PAI-1抗原和VEGF增加至接近正常水平。
PAI-1反义RNA可有效阻断PAI-1蛋白的翻译过程，并抑制主动脉平滑肌细胞中VEGF的表达。
