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目的以缺氧诱导因子1/缺氧反应元件(HIF1/HRE)基因调节系统为载体,构建含HRE启动子的人反义血管内皮生长因子(VEGF)165cDNA真核表达载体,探讨其对缺氧条件下骨肉瘤细胞VEGF表达的靶向性抑制作用。方法采用聚合酶链反应(PCR)和DNA重组技术构建由HRE启动子驱动的含荧光素酶(Luc)报告基因和人反义VEGF165cDNA全长的真核表达质粒,将其转染骨肉瘤细胞系MG63,用液闪计数仪测定缺氧对报告基因表达的调节,酶链免疫吸附试验(ELISA)法检测缺氧条件下细胞VEGF表达的变化。结果成功构建了由HRE启动子驱动的含Luc和反义VEGF165cDNA全长的真核表达质粒pBIHRELucAsVEGF165,该质粒转染骨肉瘤细胞系,缺氧条件下可使报告基因在肿瘤细胞中的表达提高3.5×102倍,使肿瘤细胞VEGF分泌下降45%。结论利用肿瘤缺氧,HRE启动子能够实现反义VEGF165的高效表达,为开展靶向性抗肿瘤血管生成治疗提供了实验依据。
Objective To construct a eukaryotic expression vector of human antisense vascular endothelial growth factor (VEGF) 165cDNA containing HRE promoter and to investigate its effect on HIF1 / HRE gene regulatory system Targeted Inhibition of VEGF Expression in Osteosarcoma Cells. Methods Eukaryotic expression plasmids containing luciferase (Luc) reporter gene and human antisense VEGF165 cDNA driven by HRE promoter were constructed by polymerase chain reaction (PCR) and DNA recombination techniques, and then transfected into osteosarcoma cell lines The expression of VEGF in MG63 cells was measured by liquid scintillation counter. The expression of VEGF was detected by enzyme linked immunosorbent assay (ELISA). Results The full-length eukaryotic expression plasmid pBIHRELucAsVEGF165 containing Luc and antisense VEGF165 cDNA driven by HRE promoter was successfully constructed and transfected into osteosarcoma cell line. Under hypoxia, the expression of reporter gene in tumor cells increased by 3.5 × 102 times, so that tumor secretion of VEGF decreased by 45%. Conclusion The hypoxia and HRE promoter can express antisense VEGF165 efficiently and provide experimental basis for targeted anti-angiogenesis therapy.