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目的 观察并比较不同前药与自杀基因HSV -tk共同作用对喉癌细胞Hep -2的体外杀伤作用。方法 构建tk基因逆转录表达载体pL(tk)SN ,在LipofectAMINETM2 0 0 0介导下转染PA3 17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 ,将重组病毒分别感染人喉癌细胞Hep -2 ,G418筛选出抗性克隆命名为Hep/tk ,以RT -PCR及Southernblot检测tk基因的整合及表达。通过观察tk基因修饰细胞生长状况及比较不同前药对喉癌细胞杀伤效应以探讨不同前药作用机理。结果 RT -PCR及Southernblot分析证明tk基因在人喉癌细胞系Hep -2中有整合及表达 ;Hep/tk与未转基因的原肿瘤细胞相比 ,二者生长速度及形态结构无明显差别 ;在不同前药敏感性试验中 ,Hep/tk显示出对GCV的高敏感性 ,而tk阳性和tk阴性细胞均显示对ACV和BVdU相对不敏感 ,tk基因修饰细胞与不同比例亲本细胞混合后经GCV治疗均显示出明显旁观者效应。结论 人喉癌细胞系Hep -2对HSV -tk/GCV系统高度敏感 ,并且有明显的旁观者效应 ,可望成为喉癌基因治疗的新途径。
Objective To observe and compare the killing effect of different prodrugs and suicide gene HSV-tk on Hep-2 cells in vitro. Methods The retroviral expression vector pL (tk) SN of tk gene was constructed and transfected into PA3 17 cells by LipofectAMINETM 2000. The recombinant plasmids were screened by G418 until resistant clones appeared, which were expanded and cultured. The virus titer was determined by NIH3T3, The viruses were respectively infected with human laryngeal carcinoma cells Hep-2 and G418. The resistant clones were selected as Hep / tk, and the integration and expression of tk gene were detected by RT-PCR and Southern blot. By observing the growth of tk gene-modified cells and comparing the killing effect of different prodrugs on laryngeal carcinoma cells, the mechanism of action of different prodrugs was discussed. Results RT-PCR and Southern blot analysis showed that tk gene was integrated and expressed in human laryngeal carcinoma cell line Hep-2. There was no significant difference in growth rate and morphology between Hep / tk and untransformed primary tumor cells. In different prodrug sensitivity assays, Hep / tk showed high sensitivity to GCV, while both tk-positive and tk-negative cells showed relatively insensitivity to ACV and BVdU. After tk gene-modified cells were mixed with different proportions of parental cells, GCV Treatment showed obvious bystander effect. Conclusion Human laryngeal carcinoma cell line Hep -2 is highly sensitive to HSV -tk / GCV system and has obvious bystander effect, which is expected to become a new way of gene therapy for laryngeal cancer.