抑制消减杂交分离肿瘤血管生成相关基因

为获得肿瘤血管生成相关基因 ,以便为抗血管形成治疗肿瘤的新策略提供有价值的靶位 ,采用人新鲜的肝癌、肺癌组织匀浆活化人脐静脉内皮细胞 (HUVEC) ,并构建了cDNA表达文库 .利用抑制消减杂交 (SSH)获得活化HUVEC高表达的基因片段 ,放射标记后筛选文库 .获得的阳性克隆进一步进行差异杂交筛选 ,去除假阳性 .共获得了 177个阳性克隆 ,对其中 74个克隆进行序列分析 ,发现它们代表 32个基因 ,其中多个与肿瘤血管生成相关 ,1个为与细胞色素c氧化酶亚单位Ⅲ同源的新基因 ,2个为功能未知的假定蛋白基因 .研究结果表明 ,用肿瘤组织匀浆模拟肿瘤内环境活化HUVEC ,并通过比较活化前后基因表达谱的差异可以分离到肿瘤血管生成相关的基因 .

Isolation of Tumor Angiogenesis Related-Genes by Suppression Subtractive Hybridization

To clone tumor angiogenesis related genes and provide valuable targets for anti angiogenesis, the gene expression profiles between human umbilical vein endothelial cells (HUVEC) activated by tumor homogenates and control HUVEC were compared. To simulate tumor endothelial cells, HUVEC was activated by tissue homogenates from liver and lung cancer. Then suppression subtractive hybridization (SSH) was performed on control HUVEC versus activated HUVEC. The differentially expressed gene fragments were obtained and labeled to screen on the activated HUVEC cDNA library consisting of 4×10 6 recombinants. The false positive clones were picked out by differential hybridization screen. The positive 177 clones were obtained, from which 80 clones were selected randomly and sequenced. Analysis of DNA sequences indicated that positive 74 clones represent 32 genes. Of the 32 genes, several associate with tumor angiogenesis and metastasis as previously reported, one is a novel gene homologous to cytochrome c oxidase Ⅲ, two are novel genes with function unknown, such as KIAA1404, MGC 3067. All these suggest that tumor angiogenesis related genes can be isolated by comparing HUVEC activated by tumor homogenates simulating tumor environment and control HUVEC.