不同核因子κB二聚体对永生化神经前体细胞存活的影响

Effects of different nuclear factor κB dimers on the survival of immortalized neural progenitor cells

目的探讨不同核因子κB（NF-κB）二聚体在缺氧缺糖条件下对永生化神经前体细胞（INPC）生存的影响。方法将含巨细胞病毒启动子的对照载体（RC／CMV）及NF-κB亚基p50、p65的真核表达载体RcCMV-p50、RcCMV-p65分别及共转染INPC。蛋白质印迹法分析不同细胞株p50及p65的表达。凝胶电泳迁移实验（EMSA）检测胞核内NF-κB的DNA结合活性。蛋白质印迹法分析胞质蛋白IκBα的表达。缺氧缺糖13h后，噻唑蓝（M丌）法测定细胞存活率。结果转基因后，共得到5株不同细胞，分别命名为INPC、INPC／CMV、INPC／p50、INPC／p65和INPC／p50p65。蛋白质印迹分析显示，p50和p65基因均能在转染相应质粒的细胞株中高效表达。EMSA表明，INPC／p50、INPC／p65和INPC／p50p65细胞胞核中分别形成p50同源二聚体、p65同源二聚体、p50p65异源二聚体和p50同源二聚体。INPC／p65和INPC／p50p65细胞株中，IκBα在胞质中表达明显升高，并且缺氧缺糖处理13h后，细胞存活率显著低于其他细胞株（Games-Howell检验，均P〈0．05）。结论p50、p65的高表达可直接导致胞核内不同NF-κB二聚体DNA结合活性的升高。在神经前体细胞中，具有转录活性的NF-κB二聚体减少了缺氧缺糖刺激后细胞的存活，但无转录活性的NF-κB二聚体并未发挥保护性作用。

Objective To investigate the effects of different nuclear factor （ NF）-κB dimers on the survival of immortalized neural progenitor cells （INPCs）. Methods The control vector RC/CMV, containing the promoter of cytomegalovirus （ CMV）, and the expression vectors, RcCMV-p50 and RcCMV-p65, containing the coding regions of NF-KB subunits p50 and p65 genes, were transfected into the INPCs by liposome respectively. Stably transfected clones were screened out following G418 selection. Subsequently, the plasmid RcCMV-p50 was transiently transfected into the INPCs which had been stably transfected with the plasmid RcCMV-p65. The expression of p50 or p65 gene was detected in each cell strain by Western blotting. And the NF-κB DNA binding activity in the cell nuclear extracts was measured by electrophoresis mobility shift assay （EMSA）. The expression of IκBa in the cytoplasm was detected by Western blotting. After oxygen and glucose deprivation for 13 h, the cell survival rate was measured by MTT assay. Results After gene transfection, five different cell strains were obtained： INPC, INPC/CMV, INPC/p50, INPC/p65, and INPC/p50p65. p50 or p65 gene was translated correctly and efficiently in the cell strains which had been transfected with the corresponding plasmids. EMSA showed that the INPC/p50, INPC/p65, and INPC/p50p65 cells all gave rise to NF-KB specific bands, which were composed of p50 homodimer, p65 homodimer, and p50 p65 heterodimer and p50 homodimer respectively. The expression of IκBa was increased significantly in the cytoplasm of the INPC/p65 and INPC/p50p65 cells. Games-Howell test showed that after oxygen and glucose deprivation for 13 h, the survival rates of the NPC/p65 and INPC/p50p65 cells were （6.0±1.0）% and （4.6±0.6）% respectively,both significantly lower than those of the INPC,INPC/CMV,and INPC/p50 cells [（72.5±6.2）%,（70.1±4.3）%,and （70.4±7.3）% respectively,all P〈0.05].Conclusions Overexpression of p50 gene and p65 gene directly enhance the DNA binding activities of different NF-κB dimers in the muclei.In neural progenitor cells,NF-κB dimers with transcriptive activity decreases the cellular survival after oxygen and glucose deprivation,but NF-κB dimers without transcriptive activity don＇t prevent the cells from death.