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目的体外、原位、体内实验观察FCU／5-氟胞嘧啶(5一FC)联合γ射线对鼻咽癌CNE-2细胞的杀伤效应,以预测其对鼻咽癌的治疗作用。方法电穿孔法将重组质粒pcDNA3．1(-) CMVe．Egr-1．FCU转染CNE-2细胞,600μg/ml G418筛选14 d获得稳定表达FCU基因的CNE-2细胞,免疫印迹法鉴定FCU基因的表达。四甲基偶氮唑盐法(MTT)检测5-FC联合不同剂量的γ射线对FCU基因表达阳性的CNE-2细胞的杀伤效应。进行原位基因治疗:取5×106对数生长期的CNE-2细胞接种裸鼠右腋下,建立鼻咽癌裸鼠移植瘤模型,接种细胞12 d后将裸鼠随机分成7组,观察不同处理对各组移植瘤生长的抑制作用。进行体内基因治疗:取5×106对数生长期表达FCU基因的CNE-2细胞接种裸鼠右腋下致瘤,接种细胞后的第5天,将裸鼠随机分成7组,观察不同处理对各组移植瘤生长的抑制作用。结果从转染细胞总蛋白中检测到相对分子质量42 000的预期蛋白。体外实验中,γ射线组细胞的相对存活率为15．85％～97．88％,而5-Fc(100μg／ml)+γ射线组细胞的相对存活率是6．58％-50．00％,两组差异有统计学意义(P<0．01);5-FC+γ射线(1．0 Gy)组细胞的相对存活率分别是2．37％～35．87％,明显低于5-FC组细胞的相对存活率12．11％～99．51％(P< 0．01)。原位基因治疗和体内基因治疗实验结果表明在5-FC或者γ射线干预下,鼻咽癌移植瘤生长不同程度受到抑制,5-FC或(和)γ射线干预组的肿瘤体积明显小于对照组或PBS干预组(P< 0．05),以5-FC联合γ射线组明显。结论FCU／5-FC联合γ射线对鼻咽癌CNE-2细胞具有杀伤作用,其有协同治疗鼻咽癌的效应。
Objective To observe the killing effect of FCU / 5-fluorocytosine (5-FC) combined with γ-rays on CNE-2 cells in vitro and in vivo in order to predict its therapeutic effect on nasopharyngeal carcinoma. Methods The recombinant plasmid pcDNA3.1 (-) CMVe was electroporated. Egr-1. CNE-2 cells were transfected with FCU, and CNE-2 cells stably expressing FCU gene were screened by 600μg / ml G418 for 14 days. The expression of FCU gene was identified by immunoblotting. The killing effect of 5-FC combined with different doses of γ-rays on CNE-2 cells with FCU gene expression was detected by MTT assay. In situ gene therapy: CNE-2 cells in logarithmic growth phase were inoculated into the right armpit of nude mice to establish a xenograft model of nasopharyngeal carcinoma in nude mice. After being seeded for 12 days, nude mice were randomly divided into 7 groups. Inhibition of different treatments on the growth of xenografts in each group. In vivo gene therapy: CNE-2 cells expressing FCU gene in 5 × 106 logarithmic growth phase were inoculated on the right axillary tumor in nude mice. On the 5th day after inoculation, nude mice were randomly divided into 7 groups. Inhibition of tumor growth in all groups. As a result, a predicted protein of 42,000 relative molecular mass was detected from the total cellular proteins transfected. In vitro experiments, the relative survival rate of γ-ray group cells was 15.85% ~ 97.88%, while the relative survival rate of 5-Fc (100μg / ml) + γ-ray group cells was 6.58% -50.00 %, The difference between the two groups was statistically significant (P <0.01). The relative survival rates of 5-FC + γ-ray (1.0 Gy) group were 2.37% ~ 35.87% The relative survival rate of 5-FC group cells was 12.11% -99.51% (P <0.01). In-situ gene therapy and in vivo gene therapy results showed that the growth of NPC was inhibited to varying degrees under the intervention of 5-FC or γ-irradiation, and the tumor volume of 5-FC or (and) γ-ray intervention group was significantly smaller than that of the control group Or PBS intervention group (P <0.05), 5-FC combined with γ-ray group was significant. Conclusions FCU / 5-FC combined with γ-ray has a killing effect on CNE-2 cells of nasopharyngeal carcinoma, which has synergistic effect on the treatment of nasopharyngeal carcinoma.