目的:观察自杀基因HSV-tk与不同细胞因子基因（IL-2、TNF-α）构建的不同融合基因表达产物对喉癌细胞系的体外杀伤作用。方法: 分别构建不同融合基因逆转录表达载体PL(TI)SN,PL(TT)SN,PL(TK)SN，在LipofectAMINETM2000介导下转染PA317包装细胞，G418筛选，直至出现抗性克隆，扩大培养，用NIH3T3测定病毒滴度，将各重组病毒分别感染人喉癌细胞系Hep-2，G418筛选出抗性克隆，分别命名为Hep/TI,Hep/TT,Hep/TK，以RT-PCR及Southern blot检测各融合基因的整合及表达。通过观察各基因修饰细胞生长状况及GCV杀伤实验鉴定融合基因的表达及杀伤效应。结果:RT-PCR及Southern blot分析证明各重组外源基因在人喉癌细胞系Hep-2中均有整合及表达。Hep/TI,Hep/TK 生长速度无明显差别，Hep/TT细胞增殖速度受到抑制。在GCV杀伤试验中，Hep/TI，Hep/TT,Hep/TK均显示出对GCV的高敏感性，其中GCV对Hep/TT的杀伤作用最为明显，各基因修饰细胞与不同比例亲本细胞混合，均显示出明显旁观者效应。结论:自杀基因与细胞因子基因共表达体系对喉癌细胞系Hep-2体外杀伤具有协同作用，并且有明显的旁观者效应，可望成为肿瘤基因治疗的新途径。

Objective: To determine if fusion genes of HSV-tk gene and cytokine gene have synergy on the cell killing of the Hep2 human laryngeal carcinoma cell line in vitro. Methods: Different fusion genes expressing vectors PL(TI)SN, PL(TT)SN and PL(TK)SN were generated by recombinant DNA technology. Hep-2 was infected by the recombinant retrovirus. The positive clones were obtained after G418 selection and were termed Hep/TI, Hep/TT and Hep/TK respectively. The integration and expression of fusion genes in Hep-2 cells were identified by RT-PCR and Southern blot. The growth state and GCV killing effect of fusion genes modified cells were used to investigate the expression of fusion genes and antitumour effect on Hep-2 cells. Results: RT-PCR and Southern blot analysis confirmed the integration and expression of fusion genes in Hep-2 cells. There was no significant difference in cell proliferation between the Hep/TI and Hep/TK,but the growth of Hep/TT was restrained. After the treatment of GCV the Hep/TI, Hep/TT and Hep/TK all showed high sensitivity to GCV. The killing effect of GCV on Hep/TT was the most siginificant and bystander effects were observed siginificantly in vitro. Conclusion:The fusion genes of HSV-tk and cytokine gene have synergistic effects on killing Hep-2 cell after treatment of GCV in vitro,which might have therapeutic potentials for laryngocarcinoma.

国家自然科学基金(39970788)资助