单纯疱疹病毒胸苷激酶基因治疗结肠癌的研究

The study on retrovirus-mediated HSV-tk/GCV suicide-gene therapy for colorectal carcinoma

目的:观察单纯疱疹病毒胸苷激酶(HSV-tk)/丙氧鸟苷(GCV)自杀基因治疗系统在体内外对结肠癌的杀伤效应。方法:构建携带 HSV-tk基因的逆转录病毒载体,将该重组逆转录病毒感染结肠癌细胞株 SW480,筛选稳定表达 tk 的细胞克隆 SW4801tk,测定 SW480/tk 细胞在体外对GCV 的敏感性;SW480/tk 细胞在裸鼠皮下成瘤,用 GCV 治疗并观察疗效。结果:HSV-tk 基因整合入 SW480细胞并在 SW480/tk 细胞中稳定表达;GCV 在体外对 SW480/tk 细胞有明显的杀伤作用。其作用呈现剂量和时间依赖性特点和旁杀伤效应,对未转染细胞则无明显毒性。裸鼠 ex vivo实验得到相应结果,治疗组肿瘤受到明显抑制。结论在 in vitra 和 ex vivo 水平,表达 HSV-tk 基因的肿瘤细胞均可被 GCV 有效杀伤,逆转录病毒介导 HSV-tk/GCV 自杀基因治疗系统有可能成为结肠癌的有效治疗方法。

The efficacy of the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) suicide-gene therapy for colorectal carcinoma was tested in vitro as well as ex vivo.Methods The recombinant retroviral vector contain- ing HSV-tk gene was constructed and SW480 colorectal carcinoma cell line was infected with the recombinant retroviurse. Following selection the SW480/tk cell line which stably expressed tk was cloned.The sensitivity of SW480/tk cells to GCV was examinated in vitro.Antitumor effects were also observed after the administration of GCV in nude mice bearing tumor derived from SW480/tk cells.Results The HSV-tk gene retrovirally mediated could inserted into genome of SW480 cells and stably expressed in SW480/tk cells.In vitro the growth inhibition studies showed that SW480/tk cells were highly sensitive to GCV in dose-depandent and time-depandent manners,with an IC50 1.5μg/ml in 72 hour,and the HSV-tk gene-modified cells SW480/tk are toxic to nearby unmodified tumor cells SW480 that were resistant to GCW, which has been termed the “bystander effect”.Ex vivo studies showed simlar results that significant tumor inhibition was found in the treatment group.Conclusion The tumor cells expressing HSV-tk gene can be eradicated by GCV in vitro as well as ex vivo.The retroviirally mediated HSV-tk/GCV suicide-gene therapy system may provide a effective approach for treatment of colorectal carcinoma.