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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 investigate the effect of vascular endothelial growth factor receptor 2 (VEGFR2) block on the inhibition of vascular endothelial growth factor (VEGF) binding to corresponding receptors in vitro and in vivo. Methods Flk 1 / KDR fragment was amplified from fetal liver by RT PCR. The fragment was recombined in PLXSN retrovirus and pET 2 8b (+) vector and expressed, purified and identified. The primary cultured mouse endothelial cells were used to observe the effect of soluble receptor protein on the growth of endothelial cells. The tumor cell lines S180 and B16 were transfected by liposome, and the in vivo biological characteristics after transfection were observed. Results The fragment of soluble VEGFR2 with a size of 100 bp was isolated from the fetal rat liver tissue on the 9th and 11th day after fertilization. The fragment was ligated to the TA cloning vector and sequenced as a fragment of the extracellular domain of VEGFR2. The soluble VEGFR2 fragment was cloned into the expression vector pET 2 8b (+). In vitro experiments show that the soluble receptor protein can effectively inhibit the growth and proliferation of endothelial cells. The soluble VEGFR2 fragment was cloned into the retroviral vector PLXSN and successfully transfected into the tumor cell lines S180 and B16. The in vivo experiments showed that the tumor weight and the volume of the transgenic cell lines were significantly reduced, and the vascular density was significantly decreased while the expression of Flk1 protein was significantly Increase. Conclusion The soluble VEGFR2 fragment is an effective bioengineering product that inhibits the formation of blood vessels and is expected to serve as a target of anti-angiogenesis gene therapy.