嘌呤核苷磷酸化酶/氟达拉滨(PNP/fludarabine)自杀基因系统对人肝癌细胞系BEL7402体外杀伤效应的研究

Study on Killing Effects of Purine Nucleoside Phosphorylase/Fludarabine (PNP/fludarabine) Suicide Gene System on Human Hepatocellular Carcinoma Cell Line BEL7402 in vitro

背景:肿瘤细胞内表达的自杀基因可将无毒性的前药转化为高毒性代谢产物以杀伤肿瘤细胞,嘌呤核苷磷酸化酶/氟达拉滨(PNP/fludarabine)自杀基因系统具有目前已知最强的旁观者效应,成为基因治疗领域中的研究热点。目的:研究PNP/fludarabine自杀基因系统对人肝癌细胞系BEL7402的体外杀伤效应。方法:应用聚合酶链反应(PCR)从大肠杆菌K12菌株基因组DNA扩增PNP基因,通过8个甘氨酸的接头克隆至真核表达载体pEGFP鄄N1。将pEGFP鄄N鄄PNP以Lipofectamine2000转染BEL7402细胞,G418筛选,直至出现抗性克隆,命名为BEL7402/PNP。应用逆转录(RT)鄄PCR和RNA印迹法(Northernblotting)鉴定PNP基因的表达。分别应用四甲基偶氮唑蓝(MTT)比色法和AnnexinV鄄FITC凋亡检测试剂盒检测PNP/fludarabine自杀基因系统对BEL7402细胞的细胞毒效应、旁观者效应和诱导凋亡作用。结果:RT鄄PCR和RNA印迹法证实转染的PNP基因能在BEL7402和BEL7402/PNP细胞中表达。低浓度fludarabine对经PNP基因转染的BEL7402细胞具有明显的细胞毒效应;旁观者效应检测显示,10%的BEL7402/PNP细胞与90%的BEL7402细胞混合,经低浓度fludarabine处理,90%的细胞可被杀死;细胞凋亡检测显示,PNP/fludarabine自杀基因系统对BEL7402细胞具有明显的诱导凋亡作用。结论:PNP/

Expression of suicide gene in tumor cells can convert nontoxic prodrugs into highly toxic metabolites to kill cancer cells; purine nucleoside phosphorylase (PNP)/fludarabine suicide gene system may have the strongest bystander effect of any enzyme/prodrug system described, and is the hot topic in present gene therapy area. Aims: To investigate the killing effects of PNP/fludarabine suicide gene system on human hepatocellular carcinoma cell line BEL7402 in vitro. Methods: The PNP gene was amplified from Escherichia coli K12 genomic DNA by polymerase chain reaction (PCR) and cloned into the eukaryotic expression vector pEGFP-N1. Between PNP gene and EGFP gene, a linker including 8 glycine was used. The BEL7402 cells were transfected with pEGFP-N-PNP mediated by Lipofectamine 2000, and screened by G418 to yield the positive clone, which was termed BEL7402/PNP. The expression of PNP gene was identified by reverse transcriptase (RT)-PCR and Northern blotting. Followed the prodrug fludarabine incubation, the cytotoxic effect, bystander effect and apoptosis induction of PNP/fludarabine suicide gene system on BEL7402 cells were detected by methyl thiazolyl tetrazolium (MTT) colorimetric method and Annexin V-FITC apoptosis detection kit, respectively. Results: RT-PCR and Northern blotting analysis confirmed the expression of PNP gene in BEL7402 and BEL7402/PNP cells. There was notable cytotoxic effect of low-dose fludarabine on BEL7402 cells transfected with PNP gene; When BEL7402/PNP cells were mixed with parental BEL7402 cells at a ratio of 10∶90, significant bystander effect was observed after incubated with low-dose fludarabine, the killing rate was 90%; Apoptosis analysis indicated that the PNP/fludarabine suicide gene system had apoptotic effect on BEL7402 cells. Conclusions: The PNP/fludarabine suicide gene system has strong killing and bystander effects on human hepatocellular carcinoma cell line BEL7402.