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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 killing effect and bystander effect of lentivirus-mediated yeast cytosine deaminase (YCD) suicide gene on human leukemia Jurkat cells. Methods: The YCD-GFP lentivirus supernatant was prepared and transfected into Jurkat cells (Jurkat / YCD-GFP cells). The expression of YCD-GFP gene was detected by flow cytometry. In vitro, the killing and bystander effects of 5-fluorocytosine (5-FC) on Jurkat / YCD-GFP cells were observed. Jurkat / YCD-GFP tumor- Cytotoxicity and Bystander Effect of FC on Transplanted Tumor Cells. The above experiments with Jukut / GFP cells as a control. Results: YCD-GFP lentivirus and Jurkat / YCD-GFP cells infected with YCD-GFP lentivirus were successfully prepared. The infection rate of YCD-GFP lentivirus to Jurkat cells was 96.93%. FCM at 232μmol / L for 72h resulted in (95.61 ± 2.07)% of Jurkat / YCD-GFP cell death, and the 5-FC content in the supernatant decreased about 80%. In vitro experiments showed that the effective target ratios of Jurkat / YCD-GFP and Jurkat cells were 1: 1:10 [(68.69 ± 4.97)% vs (97.87 ± 1.11)%, P <0.05) ± 0.27) × 105vs (3.00 ± 0.16) × 105, P <0.01]. In vivo experiments showed that the YCD / 5-FC suicide gene system also had a strong killing effect and bystander effect on Jurkat / YCD-GFP tumor-bearing mice (P <0.05). CONCLUSION: Lentivirus-mediated YCD / 5-FC suicide gene system has significant killing effect and bystander effect on human Jurkat leukemia cells.