选择复制性腺病毒对人黑色素瘤细胞的特异性杀伤作用

目的：构建靶向人黑色素瘤的选择复制性腺病毒,观察其对人黑色素瘤细胞的特异性杀伤作用。方法：以PCR克隆的方法分别获得腺病毒E1区、鼠酪氨酸酶启动子和增强子DNA片段;用基因重组的方法构建靶向人黑色素瘤的选择复制性腺病毒的穿梭载体;用同源重组的方法获得靶向人黑色素瘤的选择复制性腺病毒Adhep E1以低剂量Adhep E1分别攻击人黑色素瘤细胞系SK-Mel-1和人肝细胞癌细胞系HepG2,观察细胞形态变化及计算存活细胞数;用RT-PCR检测E1A的表达,以证实AdhepE1的特异性复制。结果：经PCR证实,获得了靶向人黑色素瘤的选择复制性腺病毒AdhepE1,人黑色素瘤细胞系SK-Mel-1对AdhepE1的裂解杀伤敏感,而人肝细胞癌细胞系HepG2则不敏感。RT-PCR的结果提示,AdhepE1在人黑色素瘤细胞中可特异地复制。结论：AdhepE1对人黑色素瘤细胞具有良好的靶向性。

The specific killing of human melanoma cells by replication selective adenovirus

Objective To construct replication selective adenovirus AdhepE1 targeting human melanoma and observe its specific killing of human melanoma cells in vitro. Methods Adenovirus E1 region, the murine tyrosinase promoter and enhancer DNA sequences were acquired respectively by PCR cloning. The shuttle plasmid of replication-selective adenovirus targeting human melanoma was constructed by DNA recombination. Replication-selective adenovirus AdhepE1 was generated by homologous recombination.The human melanoma cell line SK-Mel-1 and hepatocellular carcinoma cell line HepG2 were attacked separately by lower dose of AdhepE1. Change of cell morphology was observed and the surviving cells were calculated. The expression of E1A was assayed by RT-PCR to verify the specific-replication of AdhepE1. Results Replication selective adenovirus AdhepE1 targeting human melanoma was acquired by PCR. Human melanoma cell line SK-Mel-1 was sensitive to oncolytic killing of AdhepE1 whereas HepG2 was little responsive. The results of RT-PCR suggested that AdhepE1 replicated specifically in human melanoma cells. Conclusion AdhepE1 can selectively kill human melanoma cells.