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目的研究γ射线与人单纯疱疹病毒胸苷激酶(HSV-tk)/丙氧鸟苷(GCV)基因治疗系统联合应用对Lewis肺癌细胞的杀伤作用及其可能机制。方法以脂质体介导法将含HSV-tk基因的真核表达质粒体外转染小鼠Lewis肺癌细胞,建立稳定表达的细胞株;RT-PCR检测细胞tk基因表达;Hoechst33258/PI荧光活染、流式细胞术检测γ射线与HSV-tk/GCV系统单独及联合作用下的细胞凋亡;集落形成法检测细胞存活曲线,反映放射敏感性变化;同时检测旁观者效应及γ射线对该效应的影响。结果HSV-tk/GCV系统对转染基因的Lewis细胞(Lewis-Hytk细胞)具有选择性杀伤作用。γ射线与HSV-tk/GCV系统联合应用可使Lewis-Hytk细胞的凋亡和坏死率显著高于二者单独作用之和。0.1μg/ml GCV可使Lewis-Hytk细胞的放射敏感性明显增高(SER=1.47)。γ射线可增强HSV-tk/GCV系统的旁观者效应,二者联合作用后的Lewis-Hytk细胞的培养液对未处理过的Lewis细胞具有杀伤作用。结论γ射线与HSV-tk/GCV系统联合应用对Lewis肺癌细胞具有协同杀伤效应。γ射线增强HSV-tk/GCV系统的旁观者效应可能是其协同效应产生的机制之一。
Objective To study the killing effect of γ-ray combined with HSV-tk / GCV gene therapy system on Lewis lung cancer cells and its possible mechanism. Methods Eukaryotic expression plasmids containing HSV-tk gene were transfected into mouse Lewis lung carcinoma cells by liposome-mediated method to establish stably expressing cell lines. The expression of tk gene was detected by RT-PCR. Hoechst33258 / PI fluorescent staining The apoptosis of HSV-tk / GCV system alone and in combination was detected by flow cytometry. The cell survival curve was detected by colony formation assay, which reflected the change of radiosensitivity. At the same time, the bystander effect and the effect of γ-rays on this effect Impact. Results HSV-tk / GCV system had selective killing effect on Lewis cells (Lewis-Hytk cells) transfected with gene. The combined application of γ-rays and HSV-tk / GCV system could significantly increase the apoptosis rate and necrosis rate of Lewis-Hytk cells compared with that of HSV-tk / GCV alone. The radiosensitivity of Lewis-Hytk cells was significantly increased at 0.1μg / ml GCV (SER = 1.47). Gamma rays enhance the bystander effect of HSV-tk / GCV system, and the combined effect of Lewis-Hytk cells in culture medium can kill untreated Lewis cells. Conclusions The combination of γ-ray and HSV-tk / GCV system has a synergistic killing effect on Lewis lung cancer cells. The bystander effect of γ-ray enhancement of the HSV-tk / GCV system may be one of the mechanisms by which synergistic effects occur.