FCU/5-氟胞嘧啶自杀基因前体药物系统联合放射治疗鼻咽癌的实验研究

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。

Combining FCU/5-FU suicide gene/prodrug system and radiation in treating nasopharyngeal carcinoma: an experimental study

OBJECTIVE: To explore the in vitro, in situ and in vivo killing effects to CNE-2 cells of human nasopharyngeal carcinoma (NPC) by FCU/5-FC system combined with gamma irradiation for predicting the treatment effect on NPC. METHODS: Plasmid pcDNA3.1(-)CMVe.Egr-1. FCU was introduced into CNE-2 cells by electroporation. The transfected cells were selected by G418 (600 microg/ml) for 14 days to yield cells expressing FCU stably. The FCU protein in transfected CNE-2 cells was tested by Western blotting. In vitro response of FCU-expressing CNE-2 cells to 5-FC or gamma irradiation, alone or in combination was detected by MTT assay. Furthermore, A NPC model was employed by inoculating CNE-2 cells in the right flank of nude mice for in situ gene therapy, and after 12 days of inoculation, those rats were randomized to seven groups, then the suppression of NPC growth in model was observed after giving different treatments. Finally, FCU-expressing CNE-2 cells were inoculating in the right flank of nude mice to generae NPC xenografts for in vivo gene therapy, and after 5-day of implantation, those rats were randomized to seven groups, then the delaying of tumour growth was observed in xenografts treated with different conditions. RESULTS: A anticipated relative molecular quality 42,000 protein was obtained from total protein of FCU-expressing CNE-2 cells. The growth of FCU-positive CNE-2 cells were inhibited by 5-FC or gamma irradiation, alone or in combination, but cells treated with both 5-FC and gamma irradiation resulted in enhanced cell killing when compared with cells treated with gamma irradiation or 5-FC alone. In vitro study showed that the relative survival rates of FCU-expressing CNE-2 cells treated with gamma irradiation were 15.85% - 97.88%, while that of gamma irradiation + 5-FC (100 microg/ml) group were 6.58% - 50.00%, and there was a significant difference (P < 0.01). The MTT results also demonstrated that the relative survival rate has a striking different (P < 0.01) between 5-FC group (12.11% - 99.51%) and 5-FC + gamma irradiation (1.0 Gy) group (2.37% - 35.87%). Not only in situ but also in vivo, potent growth inhibition on the explanted NPC tumours was observed in mices treated with 5-FC or gamma irradiation, alone or in combination, among which interference of both 5-FC and gamma irradiation leaded to most distinct suppression of tumour growth. Tumour volumes in groups interfered by 5-FC and or gamma irradiation were extinctly different with the control group and PBS treatment group (P < 0.05). CONCLUSION: CNE-2 cells or nasopharyngeal carcinoma xenographs could be killed by FCU/5-FC suicide gene prodrug system or gamma irradiation, and there is a synergistic therapeutic effect on NPC between FCU/5-FC and gamma irradiation.