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目的观察双功能融合基因/5-氟胞嘧啶(FCU1/5-FC)系统对胰腺癌BxPC 3细胞系的杀伤作用。方法在脂质体的介导下将含有FCU1自杀基因的真核表达载体pcDNA3.1/FCU1导入BxPC 3细胞系,经C418筛选后获得稳定表达FCU1基因的BxPC-3/FCU1细胞株。用逆转录-聚合酶链式反应(RT-PCR)法检测FCU1基因的表达。给予前体药物5-氟胞嘧啶(5-FC)后,用噻唑蓝比色法(MVF)法测定转染前后BxPC-3细胞的存活率;流式细胞仪分析转染前后细胞周期及凋亡改变;将BxPC 3/FCU1与未转染基因的BxPC-3细胞按不同的比例混合,观测5-FC对BxPC 3细胞的旁观者效应。结果FCU1基因在BxPC-3细胞中可稳定表达;5-Fc按10、30、50、70、110、130 mg/ L处理BxPC 3/FCU1细胞后,细胞存活率依次为(97.30±0.17)%、(82.50±0.21)%、(71.20±0.35)%、(59.30±0.25)%、(35.40±0.19)%、(19.90±0.32)%、(19.20±0.17)%,与未转染组比较有统计学意义(P<0.01)。在混和细胞中,当BxPC-3/FCU1细胞所占比例分别为0、10%、20%、50%、70%、100%时,5-FC对混合细胞的杀伤率依次为0、18.9%、47.2%、76.9%、87.6%、94.3%。结论FCU1/5-FC自杀基因系统对胰腺癌细胞具有明显的杀伤作用,其旁观者效应明显。
Objective To observe the killing effect of bifunctional fusion gene / 5-fluorocytosine (FCU1 / 5-FC) on pancreatic cancer BxPC 3 cell line. Methods The eukaryotic expression vector pcDNA3.1 / FCU1 containing FCU1 suicide gene was introduced into BxPC3 cell line under the mediation of lipofectamine. After being screened by C418, BxPC-3 / FCU1 cell line stably expressing FCU1 gene was obtained. The expression of FCU1 gene was detected by reverse transcription-polymerase chain reaction (RT-PCR). After administration of 5-fluorocytosine (5-FC), the survival rate of BxPC-3 cells before and after transfection was determined by MTT method. The cell cycle and apoptosis BxPC-3 cells were transfected with BxPC-3 cells in different proportions to observe the bystander effect of 5-FC on BxPC-3 cells. Results The FCU1 gene was stably expressed in BxPC-3 cells. The viability of BxPC 3 / FCU1 cells treated with 5-Fc at 10, 30, 50, 70, 110 and 130 mg / L was 97.30 ± 0.17% (82.50 ± 0.21)%, (71.20 ± 0.35)%, (59.30 ± 0.25)%, (35.40 ± 0.19)%, (19.90 ± 0.32)% and (19.20 ± 0.17)% Statistical significance (P <0.01). In the mixed cells, when the percentage of BxPC-3 / FCU1 cells was 0, 10%, 20%, 50%, 70%, 100% respectively, the killing rates of mixed cells by 5-FC were 0, 18.9% , 47.2%, 76.9%, 87.6%, 94.3%. Conclusion FCU1 / 5-FC suicide gene system has obvious killing effect on pancreatic cancer cells, and its bystander effect is obvious.