目的: 构建由AFP基因增强子启动子调控，并携带自杀基因TK的新型条件复制型腺病毒载体，观察该载体的选择复制能力、溶瘤作用及其与前药GCV联合处理对肝癌细胞的杀伤作用。方法:以HepG2基因组DNA为模板，PCR扩增AFP基因启动子（AFPp）和增强子(AFPe)，构建表达质粒pAFPpEGFPluc和pAFPepEGFPluc，再构建由AFPep调控E1A表达、并携带TK基因的穿梭质粒pDC311AFPepE1A/CMVTK，利用AdMax系统包装腺病毒Ad.AFPepE1A/CMVTK；利用Western blotting、病毒增殖测试、细胞病变效应实验、病毒联合前药GCV对肝癌细胞的杀伤实验等鉴定病毒的复制能力、溶瘤作用和对肝癌细胞的杀伤作用。结果：成功构建的腺病毒载体 Ad.AFPepE1A/CMVTK在AFP阳性细胞中选择性复制，病毒自身具有一定的溶瘤作用；该病毒载体联合GCV前药系统处理肝癌细胞后，AFP阳性肝癌HepG2细胞和Hep3B细胞的存活率分别为(10.35±1.07)%、(15.49±5.80)%，AFP阴性的张氏肝细胞和人肺癌NCIH460细胞的存活率分别为(73.55±436)%、(74.54±9.89)% (P<0.01)。结论:构建的新型溶瘤腺病毒载体具有选择性杀伤肝癌细胞的能力，在肝癌治疗方面有良好的应用前景。

Objective: To construct a novel conditional duplicate adenovirus (CRA) regulated by AFP enhancer, AFP promoter and TK suicide gene, and to observe its duplicate abilities, cytopathic effects and cytotoxity against hepatocarcinoma cells when combined with predrug GCV.Methods: AFP gene promoter and enhancer were amplified using PCR from the genome DNA of HepG2 cells; pAFPpEGFPluc and pAFPepEGFPluc plasmids were constructed; pDC311AFPepE1A/CMVTK shuttle plasmid expression E1A gene was then constructed under the control of AFP promoter, AFP enhancer (AFPep) and TK gene. Ad.AFPepE1A/CMVTK vector was constructed by AdMax system. ElA expression, cytopathic effects and cytotoxity against hepatocarcinoma cells of Ad.AFPepE1A/CMVTK were evaluated by Western blotting, flow cytometry, CCK8 assay, respectively. Results: Ad.AFPepE1A/CMVTK was successfully constructed, which could selectively replicate in AFP positive HepG2 cells and specifically inhibit proliferation of AFP positive cells. Survival rates of AFP positive HepG2 and Hep3B cells after treated with combination of Ad.AFPepE1A/CMVTK and predrug GCV were (10.35±1.07)% and (15.49±5.80)%, respectively, which were significantly lower than those of AFP negative Chang liver cells and lung cancer NCIH460 cells (\[73.55±4.36\]% and \[74.54±9.89\]%, respectively, P<0.01). Conclusion: The constructed Ad.AFPepE1A/CMVTK, when combines with TK/GCV suicide gene system, can specifically kill AFP positive hepatocarcinoma cells, which may have a future in genebased therapy against hepatocarcinoma.

国家重点基础研究计划（973）资助项目 (No.2004CB518804);国家自然科学基金杰出青年项目 (No.30325043)