反义核酸技术对人血管内皮细胞的改造作用

Reconstruction of Human Vascular Endothelial Cells with Antisense Nucleic Acid Technology

为降低在血栓形成早期血管内皮细胞分泌的血管性血友病因子(vWF)对凝血级联反应的触发效应,利用反义核酸技术构建降低vWF表达量的血管内皮细胞.人工合成vWF部分反义核酸(pvWF)片段,构建重组的真核表达载体pcDNA3.1(+)/pvWF.测序正确后用脂质体介导的方法转染血管内皮细胞,硫酸新霉素加压筛选后扩大培养转基因细胞,RT-PCR法检测转基因细胞中vWF mRNA的表达水平.结果表明,构建的vWF反义核酸真核表达载体能有效抑制细胞中vWF mRNA的表达,为新型抗凝血材料的研究提供一种基因改造的内皮细胞.

In early stages of the formation of thrombus, the von Willebrand factor（vWF） secreted by vascular endothelial cells-connects the platelet and the wounded site of inner wall of the vascellum and then originates the cascade reaction of blood coagulation. Vascular endothelial cells in which the expression of vWF is inhibited effectively can be constructed using antisense nucleic acid technology. An antisense nucleic acid fragment of vWF was artificially synthesized and then subcloned into eukaryotic expression plasmid pcDNA3.1（＋）in the reverse direction. The plasmid pcDNA3.1（＋）/pvWF was transfected by lipofectamine into vascular endothelial cells after being sequenced to confirm the correc- tion of gene. Transgenic cells were cultured in large scale after the selection using neomycin solution. Method of RT-PCR was used to investigate the vWF mRNA expression of transgenic cells. The result showed that the eukaryotic expression plasmid of vWF antisense nucleic acid could inhibit the vWF mRNA expression effectively. So a vascular endothelial cells model was set up that can express vWF antisense nucleic acid and the work provided a genetic reforming cell for the research of anticoagulated blood materials.