肝癌特异性血管抑制疗法的真核表达载体的构建及其表达  

Construction and Expression of Hepatocellular Carcinoma-Specific Expressing Eukaryotic Vector for Anti-Angiogenesis Therapy

作　　者：徐宏勇[1] 徐立[2] 高建宏[1] 李开宗[3] 窦科峰[3] 

机构地区：[1]解放军第451医院肝胆普外科,西安710054 [2]第四军医大学西京医院消化病研究所,西安710054 [3]第四军医大学西京医院肝胆外科,西安710032

出　　处：《中国普外基础与临床杂志》2007年第1期15-18,共4页Chinese Journal of Bases and Clinics In General Surgery

基　　金：陕西省自然科学基金资助项目(编号:2001SM38)~~

摘　　要：目的构建受甲胎蛋白(AFP)增强子和白蛋白(Alb)启动子调控的含有血管抑制素(angiostatin)K5序列的真核表达载体,通过基因转染,将angiostatinK5基因导入肝癌细胞,观察angiostatinK5的表达。方法用RT-PCR法从正常人真核细胞扩增出angiostatinK5基因,将AFP增强子和Alb启动子调控序列定向克隆入真核表达载体pcDNA3.1,并将angiostatinK5cDNA置于上述调控序列之下,从而构建得到重组真核表达载体pcDNA3.1-AFAB-angiostatinK5-His。利用细胞培养、脂质体介导的细胞转染,将angiostatinK5基因导入肝癌细胞。利用SDS-PAGE和Westernblot法检测肝癌细胞中angiostatinK5的表达。结果通过酶切鉴定及DNA测序证实目的真核表达载体pcDNA3.1-AFAB-angiostatinK5-His与预期的结果相同。SDS-PAGE及Westernblot分析证明,angiostatinK5在肝癌细胞中得以表达。结论构建的肝癌特异性重组真核表达载体可增加对AFP阳性肝癌细胞的治疗的特异性,并用于实现对肝癌的特异性血管抑制作用。Objective To construct a mammalian vector encoding angiostatin kringle 5 （K5） under the control of α-fetoprotein （AFP） enhancer and albumin promoter, and to observe the expression of angiostatin by introducting angiostatin gene into hepatocellular carcinoma cells through gene transfection. Methods Angiostatin cDNA was amplified from normal human eukaryotic cells by using RT-PCR. Meanwhile, AFP enhancer and albumin promoter sequences were directed cloned and were inserted into vector pcDNA3. 1. The recombinant vector of pcDNA3. 1- AFAB-angiostatin K5-His was constructed, which contained the angiostatin K5 cDNA sequence that was under the control of the AFP enhancer and promoter. Angiostatin K5 cDNA was introduced into human AFP positive hepatocellular carcinoma cell lines with the transfected cultured cells that were mediated with Lipofectamine 2000. The expression of angiostatin K5 was analyzed by Western blot and the protein was dectected with anti-His antibody. Results The 500-base pair of angiostatin K5 was in accordance with the expected sequence and the recombinant vector of pcDNA3.1-AFAB-angiostatin K5-His was also confirmed as the anticipated sequence. The expression of angiostatin K5 in AFP positive hepatocellular carcinoma cells was detected both by SDS-PAGE and Western blot. Conclusion Efficient construction and expression of angiostatin K5 to AFP positive cells make it possible for antiangiogenesis therapy of human hepatocellular carcinomas, which may provide a promising approach.

关 键 词：肝细胞癌 血管抑制素 甲胎蛋白增强子 

分 类 号：R735.7[医药卫生—肿瘤]