慢病毒介导YCD/5-FC自杀基因系统对白血病Jurkat细胞杀伤和旁观者效应

Cytotoxicity and bystander effect of lentiviral vector-mediated YCD/5-FC suicide gene system against leukemia Jurkat cells

目的:探讨慢病毒介导的酵母菌胞嘧啶脱氨酶(yeast cytosine deaminase,YCD)自杀基因对人白血病Jurkat细胞的杀伤作用和旁观者效应。方法:制备YCD-GFP慢病毒上清,转染Jurkat细胞(即Jurkat/YCD-GFP细胞),流式细胞术检测YCD-GFP基因的表达。体外观察前体药物5-氟胞嘧啶(5-fluorocytosine,5-FC)对Jurkat/YCD-GFP细胞的杀伤情况和旁观者效应;建立Jurkat/YCD-GFP荷瘤小鼠模型,体内观察5-FC对移植瘤细胞的杀伤作用和旁观者效应。以上实验均以Jukut/GFP细胞为对照。结果:成功制备YCD-GFP慢病毒和感染YCD-GFP慢病毒的Jurkat/YCD-GFP细胞,YCD-GFP慢病毒对Jurkat细胞的感染率达96.93%。232μmol/L的5-FC作用72h可使(95.61±2.07)%的Jurkat/YCD-GFP细胞死亡,且上清中5-FC含量下降80%左右。体外实验显示,Jurkat/YCD-GFP和Jurkat细胞效靶比1∶10混合培养[(68.69±4.97)%vs(97.87±1.11)%,P<0.05)]和Transwell1∶25分层培养[(1.46±0.27)×105vs(3.00±0.16)×105,P<0.01]均存在旁观者效应。体内实验显示,YCD/5-FC自杀基因系统对Jurkat/YCD-GFP荷瘤小鼠亦具有较强的杀伤作用和旁观者效应(P<0.05)。结论:慢病毒介导的YCD/5-FC自杀基因系统对人Jurkat白血病细胞具有显著的杀伤作用和旁观者效应。

Objective：To investigate the cytotoxicity and bystander effects of lentiviral vector-mediated yeast cytosine deaminase （YCD） suicide gene against the human leukemia Jurkat cells. Methods：YCD-GFP lentiviral vector was prepared and transfected into Jurkat cells （Jurkat/YCD-GFP cells）. The expression of YCD-GFP gene was examined by flow cytometry. The cytotoxicity and bystander effects of pro-drug 5-fluorocytosine （5-FC） on Jurkat/YCD-GFP cells were measured in vitro. Nude mice bearing-Jurkat/YCD-GFP cell tumors were established and the cytotoxicity and bystander effects of 5-FC on tumor cells were measured in vivo. Results：YCD-GFP lentiviral vector and Jurkat/YCD-GFP cells （Jurkat cells infected by YCD-GFP lentiviral vector） were successfully prepared; the infection rate of YCD-GFP lentiviral vector on Jurkat cells was 96.93%. 5-FC （232 μmol/L for 72h） killed （95.61±2.07）% of Jurkat/YCD-GFP cells,and 5-FC level in the culture supernatant decreased by about 80%. Bystander effect of Jurkat/YCD-GFP cells could be observed both in mixed culture （effect：target=1：10,[68.69± 4.97]% vs [97.87±1.11]%,P〈0.05） and in Transwell culture （effect：target=1：25,[1.46±0.27]×105 vs [3.00±0.16]×105,P〈0.01）. In vivo experiment results demonstrated that YCD/5-FC suicide gene system had strong cytotoxicity and bystander effects in Jurkat/YCD-GFP tumor bearing mice （P〈0.05）. Conclusion：The lentiviral vector-mediated YCD/5-FC suicide gene system has marked cytotoxicity and bystander effects against human leukemia Jurkat cells.