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目的 从原代培养的人脐静脉血管内皮细胞 (HUVEC)提取细胞总RNA ,采用逆转录PCR(RT PCR)方法得到VEGF受体KDR全长胞外区cDNA片段 ,检验其体内抗肿瘤血管生成的作用。方法 将获得的受体基因克隆到AAV基因治疗载体pSNAV中 ,得到重组质粒pSNAV/KDR。重组质粒转染BHK细胞 ,加入辅助病毒后 ,得到表达目的蛋白的重组AAV。重组AAV表达的KDR具有与VEGF结合的活性 ,重组AAV感染人膀胱癌EJ细胞 ,皮下注射Balb c裸鼠。Ⅷ因子免疫组化染色进行微血管密度测定。结果 重组AAV感染人膀胱癌EJ细胞形成的肿瘤血管化程度明显低于对照组。结论 重组AAV介导的KDR胞外区基因可有效的抑制裸鼠人膀胱癌组织的新生血管生成
Objective To extract total cellular RNA from primary cultured human umbilical vein endothelial cells (HUVECs) and obtain cDNA fragments of full length extracellular domain of KDR by reverse transcription PCR (RT PCR), and to examine the anti-tumor angiogenesis in vivo effect. Methods The obtained receptor gene was cloned into AAV gene therapy vector pSNAV to obtain recombinant plasmid pSNAV / KDR. Recombinant plasmids were transfected into BHK cells, and after addition of the helper virus, a recombinant AAV expressing the target protein was obtained. Recombinant AAV expressed KDR with VEGF binding activity, recombinant AAV infection of human bladder cancer EJ cells, subcutaneous injection of Balb c nude mice. Ⅷ factor immunohistochemical staining of microvessel density. Results The degree of tumor vascularization induced by recombinant AAV in human bladder cancer EJ cells was significantly lower than that of the control group. Conclusion Recombinant AAV-mediated extracellular domain of KDR gene can effectively inhibit the neovascularization of human bladder cancer tissue in nude mice