可溶性血管内皮生长因子受体2片段的克隆、表达及其在肿瘤血管形成中的作用

Cloning,expression of soluble VEGFR2 fragment and its effect on tumor angiogenesis

目的 对可溶性血管内皮生长因子受体 2 (VEGFR2 )片段阻断血管内皮细胞生长因子(VEGF)与相应受体结合抑制血管形成的作用进行体内外实验研究。方法 应用RT PCR技术 ,从胎鼠肝脏扩增Flk 1/KDR片段 ,重组于逆转录病毒载体PLXSN和表达载体pET 2 8b(+) ,并行表达、纯化和鉴定。以原代培养的小鼠内皮细胞 ,观察可溶性受体蛋白对内皮细胞生长的影响。以脂质体法转染肿瘤细胞系S180和B16 ,观察基因转染后的体内生物学特点。结果 在受精后第 9,11天的胎鼠肝组织中分离出 10 0 0bp大小的可溶性VEGFR2片段 ,连接TA克隆载体 ,经测序此片段为VEGFR2胞外段部分序列。将可溶性VEGFR2片段克隆入表达载体pET 2 8b(+) ,体外实验显示 ,可溶性受体蛋白能有效抑制内皮细胞的生长和增殖。将可溶性VEGFR2片段克隆入逆转录病毒载体PLXSN并成功转染肿瘤细胞系S180和B16 ,体内实验显示 ,转基因细胞系的瘤重减轻 ,体积明显缩小 ,且其血管密度明显降低 ,而Flk1蛋白表达明显增高。结论 可溶性VEGFR2片段是一种有效的抑制血管形成的生物工程产品 ,有望作为抗血管形成基因治疗的靶点。

Objective To study the anti tumor angiogenesis effect of soluble VEGF receptor fragment by blocking the combination of VEGF and its receptor in vivo and in vitro. Methods RT PCR technique was used to amplify Flk 1/KDR fragment from embryo mouse liver, which was recombinated to expression vector pET 28b(+) and retrovirus vector PLXSN, which was induced to be expressed, purified and identified with EcoR Ⅰ and Hind Ⅲ. Mouse endothelial cells were separated, cultured and identified by immunocytochemistrical staining using Ⅷ factor related antigen antibody. The expressed product was analyzed about its effect on endothelial cell’s growth in vitro with MTT method. The retrovirus vector was transfected to tumor cell lines S180 and B16 by liposome method to observe the biological specificity in vitro after gene transfection. Results 1000 bp size sVEGFR fragment was amplify from E9, E11 embryo mouse liver tissues, which was recombinated to TA clone vector and identified by sequence analysis. This fragment was cloned to expression vector pET 28b(+), the expressed product was purified and identified correctly. The in vitro study showed this expressed product can effectively inhibit endothelial cell ,s growth and proliferation. The fragment was then cloned to retrovirus vector PLXSN and transfected to tumor cell lines S180 and B16 successfully with RT PCR and SDS PAGE. The experiments in vivo showed that the weight of tumor smaller, the size decreased significantly, the microvessel density was fewer and Flk1 protein expression were higher in the group of gene transfection than that of control. Conclusion Soluble VEGFR fragment is a kind of effective gene engineer product for anti tumor angiogenesis gene therapy and the development of anti tumor drug.