目的探讨NF-κB在永生化神经前体细胞凋亡中的作用。方法永生化神经前体细胞接种于6孔板,随机分为5组,每组6孔,INPC组不进行基因转染,INPC/CMV组转染表达质粒RcCMV;INPC/050组、INPC/p65组分别转染含050基因和p65基因的表达质粒RcCMV,转染后2d,加入200μg/ml G418筛选12~14d,进行阳性克隆扩大培养3—4周,检测050mRNA、p65mRNA表达、NF-κB活性和细胞凋亡情况。INPC/p50#5组转染含p65基因的表达质粒RcCMV,加入200μg/mlG418筛选12—14d,进行阳性克隆扩大培养3~4周,转染含pSO基因的表达质粒RcCMV,2d后进行上述指标的检测。结果INPC/050组和INPC/p50#5组p50mRNA表达高于其他组(P〈0.05),INPC/p65组和INPC/p50p65组p65mRNA表达、NF-κB活性及细胞凋亡率高于其他组(P〈0.05)。结论NF-κB活性升高可促讲永牛化神绎前体细胞的凋产.
Objective To investigate the role of NF-κB in apoptosis of immortalized neural progenitor cells ( INPCs). Methods INPCs were cultured in 6-well plates and were randomly divided into 5 groups ( n = 6 each) : group I was not transfected with any plasmid (group INPC); group II was transfected with control plasmid (group INPC/CMV) ; group m was transfeeted with plasmid RcCMV-p50 ( group INPC/p50) ; group IV was transfected with plasmid RcCMV-p65 (group INPC/p65) and groupV was transfected with plasmid RcCMV-p50 and RcCMV-p65 (group INPC/p50p65). Group INPC/CMV (II), INPC/p50 (m) and INPC/p65 (IV) were screened by G418, and the positive clones were then cultured for 3-4 weeks. The transcription of p50 mRNA or p65 mRNA was detected by RT-PCR. The NF--κB activity was measured by luciferase reporter gene assay. The cell apoptosis was measured by mmexin V/PI staining. In group INPC/p50p65 and group INPC/p65, the cultured positive clone was transiently transfected with plasmid RcCMV-p50. Two days after transfection, the same measurement was performed in group INPC/p50p65 and the other groups. Results The expression of p50 mRNA was significantly increased in group INPC/p50 and INPC/p50p65 as compared with the other groups (P 〈 0.05). The expression of p65 mRNA, the NF-κB activity and the apoptotic rate were significantly increased in group INPC/p65 and INPC/p50p65 as compared with the other groups ( P 〈 0.05). Conclusion Enhanced NF-κB activity can increase immortalized neural progenitor cell apoptosis.