重组人血管内皮生长因子D诱导鸡胚尿囊膜血管增生_医学论文.docVIP

重组人血管内皮生长因子D诱导鸡胚尿囊膜血管增生_医学论文 重组人血管内皮生长因子D诱导鸡胚尿囊膜血管增生_医学论文 作者：陈浩　丁秀云　高媛　柳晓兰　高建恩　孙启鸿【摘要】 　　为了表达和纯化具有生物学活性的人血管内皮生长因子(vascular endothelial growth factor，VEGF)，探讨VEGF对血管增生作用，从人胎肺组织中提取总RNA，设计特异性引物并应用RT法扩增VEGF成熟肽片段；经测序证明目的片段序列正确后，构建重组表达质粒pGEX并在大肠杆菌BL21（DE3）中进行GST融合蛋白的表达。结果表明，融合蛋白的分子量约为38 kD，表达量占菌体总蛋白的15%以上，抗GST和抗VEGF的抗体均能识别融合蛋白；纯化的GST融合蛋白与VEGFR具有结合活性，并能刺激红白血病细胞系HEL细胞生长；在鸡胚尿囊膜实验中GST具有促进鸡胚尿囊膜血管生成的作用。结论：成功表达了具有生物学活性的重组人VEGF融合蛋白，为进一步研究VEGF的作用机理提供了实验模型。 【关键词】 血管内皮生长因子D；血管内皮生长因子受体3；鸡胚尿囊膜；血管生成 　　 Recombinant Human VEGF Abstract Vascular endothelial growth factorD (VEGFD) and vascular endothelial growth factor receptor2,3 (VEGFR2,3) with their corresponding signaling pathway play significant roles in the development of the embryonic vascular system and pathological lymphangiogenesis. The study was aimed to express and purify the GSTVEGFD fusion protein, and to explore the angiogenesis effect of VEGFD. The total RNA was extracted from human fetal lung tissue, and the mature form of VEGFD was expanded by polymerase chain reaction (PCR), then the plasmid pGEX5X1/VEGFD was reconstructed and the GSTVEGFD fusion protein expressed in transformed E.coli BL21DE3.The results showed that the molecular mass of this fusion protein was 38 kD and compassed more than 15% of the total bacteria proteins.The fusion protein was recognized by antiGST and antiVEGFD antibodies.The soluble GSTVEGFD fusion protein could interact with VEGFR3/Fc and was able to stimulate the proliferation of human erythroleukemia cell line（HEL） cells. The data of chick embryo chorioallantoic membrane (CAM) experiments indicated that GSTe GSTVEGFD fusion protein with biological activity was successfully expressed, and which may provide an experimental model for the investigation of the VEGFDinduced angiogenesis and lymphangiogenesis. Key words VEGFDVEGFR3 angiogenesis chick embnyo chorioallantoic menbreme 肿瘤转移通常是人类癌症致死的主要原因之一。目前认为，肿瘤转移的通路主要有以下三种方式：第一，肿瘤细胞浸润血管，直接进入血道形成转移灶；第二，通过侵入已存在的淋巴管进入淋巴循环系统