The Role Of Nuclear Factor-κB Subunits P50 And P65 In The Survival Of Immortalized Neural Progenitor Cells And The Underlying Mechanism

Background and objectiveIschemic-hypoxic brain damage is one of the most common diseases that threaten the health of human. Because the ischemia and hypoxia induce the loss of neural tissue, the traditional drug treatment method is not satisfied. The neural stem cells or progenitor cells bring the hope for the repair and regeneration of the brain tissue. Neural stem cells are functionally defined as an immature cell with the capacity for self-renewal, proliferation and multi-potential, generating the main classes of neural cell types (glia and neurons). The potential of these cells suggests that they may play an important role not only in development of nervous system but also in cell replacement for maintaining the normal brain or repairing injured brain. In the ischemic-hypoxic brain damage, the endogenous migrated or exogenous transplanted neural stem cells are all under the ischemic or hypoxic microenvironment. The cell survival count is too low, or the survival time is too short to have an effective repair in the damaged brain. The nuclear factor-κB, NF-κB, in mammals, consists of five members: RelA(p65), RelB, c-Rel, p105/p50 and p100/p52, which can regulate the expression of a series of genes. The p65 and p50 proteins are considered as the most important two subunits. NF-κB is widely expressed in the nervous system. A controversy exists in the fact that the activation of NF-κB in the peri-lesion zones of cerebral ischemia is illustrated to be involved in promoting neural cell apoptosis as well as survival. All these phenomena have attracted our attention in regard to the role of increased NF-κB activity in the survival of neural progenitor cells. And could the intervention of NF-κB activity be a novel therapeutic strategy for improving the effect of neural progenitor cells for neural regeneration? We previously have established an immortalized neural progenitor cell strain (INPC) by transfection of the simian virus 40 large T antigen gene into the primary cultured neural progenitor cells of newborn rat. The purpose of this study is to explore the potential role of NF-κB in the survival of the immortalized neural progenitor cell strain and the relative mechanism by NF-κB subunits p50, p65 gene transfection. These researches will certainly help us to understand the role of NF-κB in the central nervous system development and the regulation of neural progenitor cell death induced by ischemia or hypoxia. And these findings will provide experimental basis for the novel therapeutic strategies for neuroprotection.Methods1. An effective method of non-viral vectors mediated exogenous genes transfection in the immortalized neural progenitor cellsThe plasmid EGFP-C1 was transfected into INPC by three different non-viral vectors respectively, including Lipofectamine 2000, TRANSfection and Sofast. The expression of EGFP and the transfection efficiency of the 3 vectors were measured at 24 h after transfection. The transfection efficiency of the vector which was the most efficient, was detected 12, 24, 48 and 72 h after transfection to determine the expression peak of EGFP. The viability of transfected and non-transfected cells was measured by trypan blue rejection test. After the plasmid EGFP-C1 was transfected into INPC, the stable cell clone designated INPC/EGFP was isolated by G418 selection. The positive rate of EGFP was observed in INPC/EGFP. And the specific molecular marker of neural progenitor cells, nestin, was detected using immunocytochemistry in INPC/EGFP. After INPC/EGFP was induced by fetal bovine serum, the cell morphology and the expression of EGFP were observed in the differentiated cells. The level of p65 expression was determined in plasmid RcCMV-p65 transiently transfected INPC mediated by Lipofectamine 2000 using immunocytochemistry.2. Construction of immortalized neural progenitor cell strain genetically modified by NF-κB subunits p50, p65 genesThe control vector RC/CMV and the expression vectors RcCMV-p50 and RcCMV-p65, containing the coding regions of NF-κB subunits p50 and p65 genes, were transfected into immortalized neural progenitor cell strain (INPC) by Lipofectamine 2000, respectively. Stably transfected clones were screened out following G418 selection. Individual clones were screened and expanded into clonal cell strains using limited dilution method. The individual clone in which the level of p50 or p65 was expressed mostly was screened out by immunocytochemistry and Western Blotting. Subsequently, plasmid RcCMV-p50 was transiently transfected into clonal cell strain which expressed p65 mostly. The transcription of Neo, p50 or p65 gene was detected by reverse transcription-polymerase chain reaction (RT-PCR). The translation of p50 or p65 gene was detected by Western Blotting.3. The formation, distribution and transcriptive activity of NF-κB dimer following transfection of NF-κB subunits in INPCThe NF-κB DNA binding activity was measured by electrophoresis mobility shift assay (EMSA) in the cell nuclear extracts from immortalized neural progenitor cell strain genetically modified by NF-κB subunits p50, p65 genes. The expression of IκBαin cytoplasm was detected by Western Blot. The content and cellular distribution of p50 or p65 were determined by an indirect immunocytochemistry study. And the NF-κB transcriptive activity was measured by luciferase reporter gene assay in each cell strain.4. The role of NF-κB in the survival of immortalized neural progenitor cells and the underlying mechanismUnder normal and oxygen and glucose deprivation for 1 h or 3 h conditions, NF-κB transcriptive activity in each cell strain was detected by luciferase assays. Under normal and oxygen and glucose deprivation for 3 h conditions, the apoptotic rate in each cell strain was determined by Annexin V assays. After oxygen and glucose deprivation for 6 h, the cell morphological changes were observed by bisBenzimide Hoechst 33342 staining, and the cell apoptotic rate was measured by flow cytometry (FCM) using PI staining only. Following oxygen and glucose deprivation for 12 h, the cell survival rate was measured by MTT assay. Each cell strain before and after 6 h OGD insult were harvested to detect bcl-2 and bcl-x, bax, p53, Fas ligand, XIAP and Actin genes expressions by western blotting. The band densities were analysis through ImageQuant TL software to obtain the Bax/Bcl-2 ratio.5. Statistical analysisData were presented as mean±standard deviation. The means among the groups were analyzed using two-way ANOVA with repeated measures and one-way ANOVA. Statistical significance was accepted if P < 0.05.Results1. The transfection efficiency of Lipofectamine 2000, TRANSfection and Sofast was (25.5±2.9)%, (4.0±1.7)%, (7.9±1.4)% respectively, at 24 h after transfection. And Lipofectamine 2000 was the most efficient. Its transfection efficiency at 12, 24, 48 and 72 h after transfection was (17.1±0.7)%, (25.5±2.9)%, (19.4±0.9)%, (15.6±1.4)%, respectively. The expression of EGFP was the highest at 24 h after transfection. The positive rate of EGFP was 95% in INPC/EGFP. And INPC/EGFP was still nestin positive. The differentiated cells presented as neurons or astrocytes, and EGFP was still found in their somas and processes. After transient transfection of plasmid RcCMV-p65 by Lipofectamine 2000, some cells presented as p65 positive staining, the positive rate was 15%.2. The individual clones of stable transfection of p65 including A12, A13, B11, C21, C22 and E2 were obtained following G418 selection and limited dilution method. All of them were positive for p65 immunostaining and western blotting, but the level of p65 expression was significantly higher in cultured C21. C21 was designated as INPC/p65. Subsequently, plasmid RcCMV-p50 was transiently transfected into INPC/p65, and the INPC/p50p65 cell strain was obtained. As the same, the individual clone of stable transfection of p50 in which the level of p50 was expressed mostly was designated as INPC/p50. The control vector Rc/CMV had been transfected into INPC/CMV determined by RT-PCR of Neo gene. p50 or p65 gene was transcripted or translated correctly and efficiently in the cell strains which had been transfected with the corresponding plasmids.3. In EMSA, INPC/p50, INPC/p65 and INPC/p50p65 all gave rise to NF-κB specific bands, which were composed of p50 homodimer, p65 homodimer, and p50 p65 heterodimer and p50 homodimer, respectively. The expression of IκBαwas increased significantly in the cytoplasm of INPC/p65 and INPC/p50p65. A strong, predominantly anti-p50 nuclear immunocytochemistry staining was obtained in INPC/p50. However, in INPC/p65 predominantly anti-p65 cytoplasm staining was obtained, and in several cells nuclear staining also could be seen. The NF-κB transcriptive activity slightly increased in INPC/p50, while obviously increased in INPC/p65 and INPC/p50p65 cell strain, more in former (all P<0.05).4. TheκB dependent luciferase activity was significantly different among the cell strains or treatment groups (control group, OGD 1 h group, OGD 3 h group). The spontaneous cell apoptosis and the deteriorating cell apoptosis under OGD condition were resulted from enhanced transfected NF-κB transcriptive activity in INPC/p65 and INPC/p50p65 cell strains by Annexin V assays. After oxygen and glucose deprivation for 6 h, the apoptotic morphological changes could be seen in all cell strains. And the apoptotic rate increased significantly in INPC/p65 and INPC/p50p65 cell strains, compared with INPC, INPC/CMV and INPC/p50 cell strains (P<0.05). After oxygen and glucose deprivation for 12 h, the survival rate was significantly reduced in INPC/p65 and INPC/p50p65 compared with other cell strains (P<0.05). Under normal and oxygen and glucose deprivation for 6 h conditions, transfected NF-κB transcriptive activity in INPC/p65 and INPC/p50p65 cell strains up regulated the level of Bax and Bcl-2 and the ratio of Bax to Bcl-2.Conclusions1. Exogenous genes can be effectively and conveniently transfected into INPC by Lipofectamine 2000. These provide a good basis for the further research on the biological characteristics of immortalized neural progenitor cells transfected by NF-κB genes.2. Immortalized rat neural progenitor cell strains genetically modified by NF-κB subuint p50 or p65 genes has been constructed successfully.3. Different NF-κB dimers could be directly found in the nuclear of the cell strains transfected with p50 or p65 genes, escaping cytoplasmic retention by endogenous IκBαproteins, resulting in increased NF-κB transcriptive activity.4. The nuclear NF-κB transcriptive activity induced by NF-κB subunits transfection results in spontaneous cell apoptosis, furthermore aggravates the cell apoptosis under OGD condition in an immortalized neural progenitor cell line, which is partly carried out through the Bcl-2 family dependent pathway.SummaryIn these researches, immortalized neural progenitor cell strains genetically modified by NF-κB subunits p50, p65 genes have been successfully constructed by liposome transfection technology. And we confirm overexpression of p50, p65 results in different NF-κB dimers in the cellular nuclear, escaping cytoplasmic retention by endogenous IκBαproteins, and leads to increased NF-κB transcriptive activity. Then we observe the upregulated NF-κB transcriptive activity results in spontaneous cell apoptosis, furthermore increases the vulnerability to injury in the immortalized neural progenitor cell. Finally, we demonstrate this is partly carried out through the Bcl-2 family dependent pathway. These researches will help us to understand the role of NF-κB during central nervous system development and in the regulation of ischemic or hypoxic neural progenitor cell death. And these findings will provide experimental basis for the novel therapeutic strategies for neuroprotection. Objective To investigate the culture, proliferation and differentiation of human neural progenitor cells derived from embryonic brains of different age or region. Methods The free-floating cells were cultured as 6-9 week embryonic whole brains group, 14-17 week embryonic whole brain group, 14-17 week embryonic neocortex group, 14-17 week embryonic striatum group, 14-17 week embryonic diencephalons group, 14-17 week embryonic mesencephalon group, 14-17 week embryonic metencephalon group and 14-17 week embryonic myelencephalon group. The expression of nestin, self-renew and multipotential property of the cell clusters was identified. The growth and proliferation of the cell aggregations of each group were observed. After the neurospheres from 6-9 week embryonic whole brains group, 14-17 week embryonic whole brain group, 14-17 week embryonic neocortex group, 14-17 week embryonic striatum group and 14-17 week embryonic diencephalons group were induced, the percentage of neuron or astrocyte was investigated by immunocytochemical staining. Results Nestin and Brdu immunochemistry staining were positive in the cell aggregations of each group. And they could differentiate into MAP2 or GFAP positive cells. One week in vitro, there were the most neurospheres in 14-17 week embryonic striatum group. And there was a decreased trend in the numbers of neurospheres of 14-17 week embryonic diencephalons group, 6-9 week embryonic whole brains group, 14-17 week embryonic whole brain group, 14-17 week embryonic neocortex group. Only few neurosphere was observed in other groups. Single cell from 14-17 week embryonic striatum group could form clone. After differentiation of neural progenitor cells from 6-9 week embryonic whole brains group, 14-17 week embryonic whole brain group, 14-17 week embryonic neocortex group, 14-17 week embryonic striatum group and 14-17 week embryonic diencephalons group, the percentage of MAP2 or GFAP positive cells have no significant difference among them. Conclusion In vitro, neural progenitor cells can be isolated from embryonic brains of different age or region. Different region should be obtained as primary culture according to the different age embryonic. Under the same culture conditions, the percentage of differentiated neuron or astrocyte has no difference in the neural progenitor cells from embryonic brains of different age or region.