Gui Ling-Li, Zhang Chuan-Han, Liu Zhi-Heng, Chen Zhao-Jun, Zhu Chang
Department of Anesthesiology, Tongji Hospital, Tongi Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Yi Xue Za Zhi. 2008 Apr 1;88(13):871-5.
To investigate the effects of different nuclear factor (NF)-KB dimers on the survival of immortalized neural progenitor cells (INPCs).
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-KB DNA binding activity in the cell nuclear extracts was measured by electrophoresis mobility shift assay (EMSA). The expression of IkappaBalpha 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.
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-kappaB specific bands, which were composed of p50 homodimer, p65 homodimer, and p50 p65 heterodimer and p50 homodimer respectively. The expression of IkappaBbeta 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].
Overexpression of p50 gene and p65 gene directly enhance the DNA binding activities of different NF-kappaB dimers in the nuclei. In neural progenitor cells, NF-kappaB dimers with transcriptive activity decreases the cellular survival after oxygen and glucose deprivation, but NF-kappaB dimers without transcriptive activity don't prevent the cells from death.
探讨不同核因子（NF）-κB二聚体对永生化神经祖细胞（INPCs）存活的影响。
分别用脂质体将含有巨细胞病毒（CMV）启动子的对照载体RC/CMV以及含有NF-κB亚基p50和p65基因编码区的表达载体RcCMV-p50和RcCMV-p65转染至INPCs。经G418筛选后筛选出稳定转染的克隆。随后，将质粒RcCMV-p50瞬时转染至已稳定转染质粒RcCMV-p65的INPCs。通过蛋白质免疫印迹法检测各细胞株中p50或p65基因的表达。采用电泳迁移率变动分析（EMSA）检测细胞核提取物中NF-κB的DNA结合活性。通过蛋白质免疫印迹法检测细胞质中IκBα的表达。氧糖剥夺13小时后，采用MTT法检测细胞存活率。
基因转染后，获得了5种不同的细胞株：INPC、INPC/CMV、INPC/p50、INPC/p65和INPC/p50p65。p50或p65基因在转染了相应质粒的细胞株中能够正确且高效地翻译。EMSA结果显示，INPC/p50、INPC/p65和INPC/p50p65细胞均出现了NF-κB特异性条带，分别由p50同源二聚体、p65同源二聚体、p50 p65异源二聚体和p50同源二聚体组成。INPC/p65和INPC/p50p65细胞的细胞质中IκBβ的表达显著增加。Games-Howell检验显示，氧糖剥夺13小时后，NPC/p65和INPC/p50p65细胞的存活率分别为（6.0±1.0）%和（4.6±0.6）%，均显著低于INPC、INPC/CMV和INPC/p50细胞[分别为（72.5±6.2）%、（70.1±4.3）%和（70.4±7.3）%，均P<0.05]。
p50基因和p65基因的过表达直接增强了细胞核中不同NF-κB二聚体的DNA结合活性。在神经祖细胞中，具有转录活性的NF-κB二聚体会降低氧糖剥夺后的细胞存活率，但无转录活性的NF-κB二聚体并不能防止细胞死亡。
