目的 建立具有绿色荧光蛋白（GFP）示踪功能的单纯疱疹病毒1型胸苷激酶基因/更昔洛韦自杀基因系统（HSV1-tkGFP/GCV），并检测该系统对小鼠黑色素瘤的抑制作用。方法 将阳性重组质粒pLXSN-tkGFP和pLXSN-DsRed2分别转染B16细胞，加G418进行筛选。用GCV（5，50，500 μmol·L-1）杀伤实验验证tk基因的表达。四甲基偶氮唑盐（MTT）法和荧光示踪法验证该系统存在旁杀伤效应。将B16-tkGFP和B16-RFP细胞按1∶1混合，接种于C57BL/6J小鼠进行肿瘤模型复制，随机分为模型组和GCV（50 mg·kg-1·d-1）组，腹腔注射给药，每3 d检测1次肿瘤大小（n=14）。结果 成功获得能发出绿色荧光的B16-tkGFP细胞和能发出红色荧光的B16-RFP细胞，且tk基因在B16-tkGFP细胞中表达正常。该系统存在一定的旁杀伤效应。与模型组比较，GCV组小鼠肿瘤生长受到明显抑制（P＜0.01）。结论 重组HSV1-tkGFP/GCV系统对小鼠黑色素瘤有明显抑制作用，并可实现直观检测其旁杀伤效应，为后续中药联合自杀基因疗法的研究奠定基础。

Objective To construct recombinant herpes simplex virus type 1 thymidine kinase/ganciclovir（HSV1- tkGFP/GCV） tumor suicide gene system with green fluorescent protein（GFP） monitoring function，and to verify the inhibitory effect of this system on murine melanoma. Methods The positive recombinant plasmid（pLXSN-tkGFP or pLXSN-DsRed2） was transfected into B16 cells and then anti-G418 monoclone cells were obtained after screening with G418.The presence and expression of tk gene in B16-tkGFP cells was confirmed by the killing effects of GCV（5，50，500 μmol·L-1）. The bystander effect of HSV1-tkGFP/GCV system was measured by MTT assay，fluorescence image method. A mixture of B16-tkGFP and B16-RFP cells at the ratio of 1∶1 was inoculated subcutaneously into C57BL/6J mice for the modeling. The tumor-bearing mice were randomly divided into tumor model group and GCV（50 mg·kg-1·d-1）treated group. GCV was administrated intraperitoneally. The tumor size was measured every three days（n=14）. Results B16-tkGFP moneclone and B16-RFP moneclone were obtained successfully. The recombinant HSV1-tkGFP/GCV system had some bystander effect. The tumor growth in the GCV treated group was significantly inhibited as compared with that in the tumor model group（P＜0.01）. Conclusion The HSV1-tkGFP/GCV system shows obvious inhibition of murine melanoma，and the bystander effect could be observed directly，which would lay the foundation for further study of HSV1-tkGFP/GCV system combining with Chinese medicine.

R285.5

国家自然科学基金资助项目（81072906，30973811）；广东省科技厅承担政府特定任务项目（2012B061700108）。