核因子-κB亚基p50和p65对永生化神经前体细胞存活的影响及机制研究
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摘要
研究背景和目的
缺血缺氧性脑损伤是严重威胁人类生命健康的常见疾病之一,其导致神经组织缺失,传统的药物治疗疗效难以令人满意。神经干细胞或前体细胞的发现为组织再生修复带来了希望。神经干细胞或前体细胞是一类具有自我更新能力、增殖能力和多向分化潜能的未成熟细胞。它在神经系统发育、正常脑功能的维护和脑损伤的修复中发挥了重要的作用。但是研究发现,由于局部缺血缺氧性微环境的改变,内源性迁移或外源性移植的神经干细胞或前体细胞存活数量少、存活时间短,难以发挥有效的修复作用。核因子-κB (NF-κB)是一种核转录因子,能调控一系列基因的表达。它由5种亚基组成,p65和p50是其中最重要的两个亚基。NF-κB在神经系统中广泛表达,在缺血状态下,缺血灶周围组织中NF-κB被激活,但其发挥保护作用还是促凋亡作用,目前尚存争议。以上这些现象使我们关注到这样一个问题,缺血灶周围NF-κB激活对神经干细胞或前体细胞的存活有无影响?干预NF-κB能否成为改善神经前体细胞脑损伤修复作用的新策略?
本课题拟以永生化神经前体细胞株(INPC)为细胞模型,通过NF-κB亚基p50、p65基因转染探讨其对神经前体细胞存活的影响及其机制,进而明确NF-κB在神经发生学和缺血缺氧性神经前体细胞损伤中的作用,为进一步探讨神经前体细胞修复治疗缺血性脑损伤奠定理论基础。
研究方法
1.介导外源基因导入永生化神经前体细胞株非病毒载体的选择
用非病毒载体阳离子脂质体Lipofectamine 2000,TRANSfection或阳离子聚合物Sofast分别负载质粒EGFP-C1转染INPC,转染后24 h检测转染效率。对转染效率最高的一种载体,观察其转染后12、24、48和72 h的转染效率,确定EGFP表达最高峰。用台盼蓝排斥试验检测转染和未转染细胞的活力。质粒EGFP-C1转染INPC后,经新霉素类似物G418筛选,挑选稳定细胞克隆INPC/EGFP,并观察其EGFP阳性率。应用巢蛋白(Nestin)抗体鉴定INPC/EGFP。胎牛血清诱导INPC/EGFP分化,观察分化后细胞的形态及EGFP表达。免疫细胞化学检测脂质体Lipofectamine 2000介导RcCMV-p65质粒瞬时转染INPC后p65的表达。
2.NF-κB亚基p50、p65基因修饰的永生化神经前体细胞株的构建
采用脂质体Lipofectamine 2000将p50和p65编码质粒RcCMV-p50、RcCMV-p65和对照空质粒Rc/CMV分别转染永生化大鼠神经前体细胞株(INPC)。经G418筛选,挑选阳性克隆。采用有限稀释法分离单细胞克隆并扩大培养。采用免疫细胞化学和免疫印迹法挑选p50或p65表达量最高的单细胞克隆。瞬时转染RcCMV-p50质粒于已挑选出的p65表达量最高的单细胞克隆。RT-PCR检测新霉素(Neo)基因、p50或p65基因转录水平的表达。免疫印迹法检测p50或p65基因蛋白的表达。
3.转染NF-κB亚基后不同二聚体的形成、分布及转录活性
将NF-κB亚基p50、p65基因修饰后的不同永生化神经前体细胞株进行凝胶电泳迁移实验(EMSA)检测胞核内NF-κB的DNA结合活性。免疫印迹分析胞浆NF-κB抑制蛋白(IκBα)的表达。免疫细胞化学法检测p50和p65分别在细胞中的含量及胞浆胞核的分布。运用荧光素酶报告系统检测各细胞株NF-κB转录活性。
4.NF-κB对永生化神经前体细胞株存活的影响及相关作用机理
在正常及缺氧缺糖1 h或3 h条件下,荧光素酶报告系统检测各细胞株的NF-κB转录活性,Annexin V和碘化丙锭( PI)双标记法流式细胞仪检测各细胞株凋亡率。缺氧缺糖6 h后,双苯甲亚胺Hoechst 33342染细胞核,观察细胞形态学改变,PI单染流式细胞术检测细胞凋亡率。缺氧缺糖12h后,四甲基偶氮唑盐比色试验(MTT试验)测定细胞存活率。在正常及缺氧缺糖6 h条件下,免疫印迹分析各细胞株NF-κB凋亡相关靶基因Bcl-2, Bax, Bcl-Xs/l, FAS-L,p53, XIAP和Actin蛋白的表达,通过Amersham公司ImageQuant TL软件分析条带的光密度值,计算各细胞株Bax/Bcl-2比值。
5.统计学分析
采用SPSS11.5统计软件分析数据,计量资料以均数±标准差(±s)表示。组间多个样本均数的比较采用重复测量的双因素方差分析或单因素方差分析,P<0.05为差异有统计学意义。
研究结果
1.Lipofectamine 2000,TRANSfection和Sofast转染INPC后24 h,转染效率分别为(25.5±2.9)%,(4.0±1.7)%,(7.9±1.4)%,Lipofectamine 2000的转染效率最高。其转染后12、24、48和72 h的转染效率分别为(17.1±0.7)%,(25.5±2.9)%,(19.4±0.9)%,(15.6±1.4)%,EGFP在转染后24 h表达最高。INPC/EGFP中,EGFP阳性率约为95%。INPC/EGFP巢蛋白表达阳性。其分化后呈神经元或星形胶质细胞样,且胞体及突起中仍可见绿色荧光。Lipofectamine 2000介导RcCMV-p65质粒的瞬时转染后,部分细胞呈p65阳性染色,阳性率约为15%。
2.通过稳定筛选和有限稀释法分离得到稳定转染p65的单细胞克隆A12、A13、B11、C21、C22和E2。其p65免疫染色和免疫印迹分析均呈阳性,但C21的p65表达强于其它克隆。将C21克隆命名为INPC/p65细胞株,进行后续实验,并再次瞬时转染RcCMV-p50质粒,获得INPC/p50p65细胞株。同样得到稳定转染RcCMV-p50、RcCMV质粒的单细胞克隆,分别命名为INPC/p50和INPC/CMV细胞株。Neo基因RT-PCR显示,对照空质粒已成功转染至INPC/CMV。RT-PCR和免疫印迹分析显示,p50和p65基因均能在稳定转染相应质粒的细胞株中高效表达。
3.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在胞浆中表达升高。免疫细胞化学显示INPC/p50细胞株中主要为强的胞核p50阳性染色,然而INPC/p65细胞株中主要为胞浆p65阳性染色,但在一些细胞中也可见胞核p65阳性染色。NF-κB转录活性在INPC/p50细胞株中轻度增高,在INPC/p65、INPC/p50p65细胞株中明显的升高,其中以INPC/p65细胞株最高(均P<0.05)。
4.κB依赖性的荧光素酶活性在不同的NF-κB亚基转染细胞株间及不同的缺氧缺糖处理组间(对照组,缺氧缺糖1 h组,缺氧缺糖3 h组)有显著性差异。Annexin V-FITC凋亡检测发现,INPC/p65和INPC/p50p65细胞株发生了自发性的细胞凋亡,而且对缺氧缺糖刺激更为敏感。缺氧缺糖6 h后,各细胞株可观察到凋亡形态学改变,流式细胞术检测证实INPC/p65和INPC/p50p65细胞株的凋亡率升高(P<0.05),并且缺氧缺糖12 h后,细胞存活率低于其它细胞株(P<0.05)。在正常及缺氧缺糖处理6 h条件下, Bax和Bcl-2蛋白的表达及两者的比值在INPC/p65和INPC/p50p65细胞株中升高(P<0.05)。
研究结论
1.Lipofectamine 2000可高效简便地转染INPC。这为探讨转NF-κB基因对永生化神经前体细胞的生物学特性影响奠定了实验基础。
2.成功构建了NF-κB亚基p50、p65基因修饰的永生化大鼠神经前体细胞株。
3.p50、p65的高表达可逃逸胞浆内内源性IκBα的阻滞作用,导致胞核内形成不同的NF-κB二聚体,并可直接升高NF-κB转录活性。
4.转染NF-κB不同亚基后升高的NF-κB转录活性导致神经前体细胞发生自发性凋亡,并对凋亡性刺激更加敏感,而这一现象可能通过Bcl-2家族依赖性途径介导。
研究总结
本课题通过脂质体基因转染技术构建了NF-κB亚基p50、p65基因修饰的永生化神经前体细胞株,并证实p50、p65的高表达可逃逸胞浆内内源性IκBα的阻滞作用,导致胞核内形成不同的NF-κB二聚体,并直接升高NF-κB转录活性,从而观察到升高的NF-κB转录活性导致神经前体细胞自发凋亡,并对凋亡性刺激更加敏感,这一现象可能通过Bcl-2家族依赖性途径介导。上述研究有助于我们明确NF-κB在神经发生学和缺血缺氧性神经干细胞或前体细胞损伤中的作用,抑制NF-κB活性可能成为改善神经前体细胞脑损伤修复作用的新策略。
目的研究人胚不同胚龄或脑区神经前体细胞(NPC)体外培养及增殖分化特性。方法取人胚脑组织原代细胞分为小胚龄全脑组、较大胚龄全脑组、较大胚龄新皮质组、较大胚龄纹状体组、较大胚龄间脑组、较大胚龄中脑组、较大胚龄后脑组和较大胚龄延髓组8组,悬浮培养。鉴定细胞球巢蛋白抗原的表达,分化及自我更新能力。观察各组培养细胞的生长、增殖状况。运用免疫荧光细胞化学法比较小胚龄全脑组、较大胚龄全脑组、较大胚龄新皮质组、较大胚龄纹状体组及较大胚龄间脑组神经球分化后,神经元及星形胶质细胞的比例。结果各组培养出的悬浮细胞球巢蛋白抗原阳性,可分化为微管相关蛋白2(MAP2)或胶质纤维酸性蛋白(GFAP)阳性细胞,且5-溴-2-脱氧尿苷(BrdU)掺入实验阳性。培养一周,较大胚龄纹状体组的神经球数目最多,形态最规则,其次分别为较大胚龄间脑组、小胚龄全脑组、较大胚龄全脑组、较大胚龄新皮质组,其它组仅见个别神经球。采用有限稀释法可从较大胚龄纹状体组挑选单细胞克隆球。小胚龄全脑组、较大胚龄全脑组、较大胚龄新皮质组、较大胚龄纹状体组及较大胚龄间脑组NPC诱导分化后,MAP2或GFAP阳性细胞率组间比较差异无显著性。结论从不同胚龄和脑区的中枢神经系统来源的人神经前体细胞均能在体外扩增,针对不同胚龄可采用不同的原代取材方法,小胚龄可取全脑组织培养,而大胚龄则可取纹状体等特定脑区进行培养。不同胚龄或脑区来源的NPC星形胶质细胞及神经元分化比例一致。
Background and objective
Ischemic-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.
Methods
1. An effective method of non-viral vectors mediated exogenous genes transfection in the immortalized neural progenitor cells
The 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 genes
The 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 INPC
The 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 mechanism
Under 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 analysis
Data 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.
Results
1. 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.
Conclusions
1. 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.
Summary
In 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.
缺血缺氧性脑损伤是严重威胁人类生命健康的常见疾病之一,其导致神经组织缺失,传统的药物治疗疗效难以令人满意。神经干细胞或前体细胞的发现为组织再生修复带来了希望。神经干细胞或前体细胞是一类具有自我更新能力、增殖能力和多向分化潜能的未成熟细胞。它在神经系统发育、正常脑功能的维护和脑损伤的修复中发挥了重要的作用。但是研究发现,由于局部缺血缺氧性微环境的改变,内源性迁移或外源性移植的神经干细胞或前体细胞存活数量少、存活时间短,难以发挥有效的修复作用。核因子-κB (NF-κB)是一种核转录因子,能调控一系列基因的表达。它由5种亚基组成,p65和p50是其中最重要的两个亚基。NF-κB在神经系统中广泛表达,在缺血状态下,缺血灶周围组织中NF-κB被激活,但其发挥保护作用还是促凋亡作用,目前尚存争议。以上这些现象使我们关注到这样一个问题,缺血灶周围NF-κB激活对神经干细胞或前体细胞的存活有无影响?干预NF-κB能否成为改善神经前体细胞脑损伤修复作用的新策略?
本课题拟以永生化神经前体细胞株(INPC)为细胞模型,通过NF-κB亚基p50、p65基因转染探讨其对神经前体细胞存活的影响及其机制,进而明确NF-κB在神经发生学和缺血缺氧性神经前体细胞损伤中的作用,为进一步探讨神经前体细胞修复治疗缺血性脑损伤奠定理论基础。
研究方法
1.介导外源基因导入永生化神经前体细胞株非病毒载体的选择
用非病毒载体阳离子脂质体Lipofectamine 2000,TRANSfection或阳离子聚合物Sofast分别负载质粒EGFP-C1转染INPC,转染后24 h检测转染效率。对转染效率最高的一种载体,观察其转染后12、24、48和72 h的转染效率,确定EGFP表达最高峰。用台盼蓝排斥试验检测转染和未转染细胞的活力。质粒EGFP-C1转染INPC后,经新霉素类似物G418筛选,挑选稳定细胞克隆INPC/EGFP,并观察其EGFP阳性率。应用巢蛋白(Nestin)抗体鉴定INPC/EGFP。胎牛血清诱导INPC/EGFP分化,观察分化后细胞的形态及EGFP表达。免疫细胞化学检测脂质体Lipofectamine 2000介导RcCMV-p65质粒瞬时转染INPC后p65的表达。
2.NF-κB亚基p50、p65基因修饰的永生化神经前体细胞株的构建
采用脂质体Lipofectamine 2000将p50和p65编码质粒RcCMV-p50、RcCMV-p65和对照空质粒Rc/CMV分别转染永生化大鼠神经前体细胞株(INPC)。经G418筛选,挑选阳性克隆。采用有限稀释法分离单细胞克隆并扩大培养。采用免疫细胞化学和免疫印迹法挑选p50或p65表达量最高的单细胞克隆。瞬时转染RcCMV-p50质粒于已挑选出的p65表达量最高的单细胞克隆。RT-PCR检测新霉素(Neo)基因、p50或p65基因转录水平的表达。免疫印迹法检测p50或p65基因蛋白的表达。
3.转染NF-κB亚基后不同二聚体的形成、分布及转录活性
将NF-κB亚基p50、p65基因修饰后的不同永生化神经前体细胞株进行凝胶电泳迁移实验(EMSA)检测胞核内NF-κB的DNA结合活性。免疫印迹分析胞浆NF-κB抑制蛋白(IκBα)的表达。免疫细胞化学法检测p50和p65分别在细胞中的含量及胞浆胞核的分布。运用荧光素酶报告系统检测各细胞株NF-κB转录活性。
4.NF-κB对永生化神经前体细胞株存活的影响及相关作用机理
在正常及缺氧缺糖1 h或3 h条件下,荧光素酶报告系统检测各细胞株的NF-κB转录活性,Annexin V和碘化丙锭( PI)双标记法流式细胞仪检测各细胞株凋亡率。缺氧缺糖6 h后,双苯甲亚胺Hoechst 33342染细胞核,观察细胞形态学改变,PI单染流式细胞术检测细胞凋亡率。缺氧缺糖12h后,四甲基偶氮唑盐比色试验(MTT试验)测定细胞存活率。在正常及缺氧缺糖6 h条件下,免疫印迹分析各细胞株NF-κB凋亡相关靶基因Bcl-2, Bax, Bcl-Xs/l, FAS-L,p53, XIAP和Actin蛋白的表达,通过Amersham公司ImageQuant TL软件分析条带的光密度值,计算各细胞株Bax/Bcl-2比值。
5.统计学分析
采用SPSS11.5统计软件分析数据,计量资料以均数±标准差(±s)表示。组间多个样本均数的比较采用重复测量的双因素方差分析或单因素方差分析,P<0.05为差异有统计学意义。
研究结果
1.Lipofectamine 2000,TRANSfection和Sofast转染INPC后24 h,转染效率分别为(25.5±2.9)%,(4.0±1.7)%,(7.9±1.4)%,Lipofectamine 2000的转染效率最高。其转染后12、24、48和72 h的转染效率分别为(17.1±0.7)%,(25.5±2.9)%,(19.4±0.9)%,(15.6±1.4)%,EGFP在转染后24 h表达最高。INPC/EGFP中,EGFP阳性率约为95%。INPC/EGFP巢蛋白表达阳性。其分化后呈神经元或星形胶质细胞样,且胞体及突起中仍可见绿色荧光。Lipofectamine 2000介导RcCMV-p65质粒的瞬时转染后,部分细胞呈p65阳性染色,阳性率约为15%。
2.通过稳定筛选和有限稀释法分离得到稳定转染p65的单细胞克隆A12、A13、B11、C21、C22和E2。其p65免疫染色和免疫印迹分析均呈阳性,但C21的p65表达强于其它克隆。将C21克隆命名为INPC/p65细胞株,进行后续实验,并再次瞬时转染RcCMV-p50质粒,获得INPC/p50p65细胞株。同样得到稳定转染RcCMV-p50、RcCMV质粒的单细胞克隆,分别命名为INPC/p50和INPC/CMV细胞株。Neo基因RT-PCR显示,对照空质粒已成功转染至INPC/CMV。RT-PCR和免疫印迹分析显示,p50和p65基因均能在稳定转染相应质粒的细胞株中高效表达。
3.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在胞浆中表达升高。免疫细胞化学显示INPC/p50细胞株中主要为强的胞核p50阳性染色,然而INPC/p65细胞株中主要为胞浆p65阳性染色,但在一些细胞中也可见胞核p65阳性染色。NF-κB转录活性在INPC/p50细胞株中轻度增高,在INPC/p65、INPC/p50p65细胞株中明显的升高,其中以INPC/p65细胞株最高(均P<0.05)。
4.κB依赖性的荧光素酶活性在不同的NF-κB亚基转染细胞株间及不同的缺氧缺糖处理组间(对照组,缺氧缺糖1 h组,缺氧缺糖3 h组)有显著性差异。Annexin V-FITC凋亡检测发现,INPC/p65和INPC/p50p65细胞株发生了自发性的细胞凋亡,而且对缺氧缺糖刺激更为敏感。缺氧缺糖6 h后,各细胞株可观察到凋亡形态学改变,流式细胞术检测证实INPC/p65和INPC/p50p65细胞株的凋亡率升高(P<0.05),并且缺氧缺糖12 h后,细胞存活率低于其它细胞株(P<0.05)。在正常及缺氧缺糖处理6 h条件下, Bax和Bcl-2蛋白的表达及两者的比值在INPC/p65和INPC/p50p65细胞株中升高(P<0.05)。
研究结论
1.Lipofectamine 2000可高效简便地转染INPC。这为探讨转NF-κB基因对永生化神经前体细胞的生物学特性影响奠定了实验基础。
2.成功构建了NF-κB亚基p50、p65基因修饰的永生化大鼠神经前体细胞株。
3.p50、p65的高表达可逃逸胞浆内内源性IκBα的阻滞作用,导致胞核内形成不同的NF-κB二聚体,并可直接升高NF-κB转录活性。
4.转染NF-κB不同亚基后升高的NF-κB转录活性导致神经前体细胞发生自发性凋亡,并对凋亡性刺激更加敏感,而这一现象可能通过Bcl-2家族依赖性途径介导。
研究总结
本课题通过脂质体基因转染技术构建了NF-κB亚基p50、p65基因修饰的永生化神经前体细胞株,并证实p50、p65的高表达可逃逸胞浆内内源性IκBα的阻滞作用,导致胞核内形成不同的NF-κB二聚体,并直接升高NF-κB转录活性,从而观察到升高的NF-κB转录活性导致神经前体细胞自发凋亡,并对凋亡性刺激更加敏感,这一现象可能通过Bcl-2家族依赖性途径介导。上述研究有助于我们明确NF-κB在神经发生学和缺血缺氧性神经干细胞或前体细胞损伤中的作用,抑制NF-κB活性可能成为改善神经前体细胞脑损伤修复作用的新策略。
目的研究人胚不同胚龄或脑区神经前体细胞(NPC)体外培养及增殖分化特性。方法取人胚脑组织原代细胞分为小胚龄全脑组、较大胚龄全脑组、较大胚龄新皮质组、较大胚龄纹状体组、较大胚龄间脑组、较大胚龄中脑组、较大胚龄后脑组和较大胚龄延髓组8组,悬浮培养。鉴定细胞球巢蛋白抗原的表达,分化及自我更新能力。观察各组培养细胞的生长、增殖状况。运用免疫荧光细胞化学法比较小胚龄全脑组、较大胚龄全脑组、较大胚龄新皮质组、较大胚龄纹状体组及较大胚龄间脑组神经球分化后,神经元及星形胶质细胞的比例。结果各组培养出的悬浮细胞球巢蛋白抗原阳性,可分化为微管相关蛋白2(MAP2)或胶质纤维酸性蛋白(GFAP)阳性细胞,且5-溴-2-脱氧尿苷(BrdU)掺入实验阳性。培养一周,较大胚龄纹状体组的神经球数目最多,形态最规则,其次分别为较大胚龄间脑组、小胚龄全脑组、较大胚龄全脑组、较大胚龄新皮质组,其它组仅见个别神经球。采用有限稀释法可从较大胚龄纹状体组挑选单细胞克隆球。小胚龄全脑组、较大胚龄全脑组、较大胚龄新皮质组、较大胚龄纹状体组及较大胚龄间脑组NPC诱导分化后,MAP2或GFAP阳性细胞率组间比较差异无显著性。结论从不同胚龄和脑区的中枢神经系统来源的人神经前体细胞均能在体外扩增,针对不同胚龄可采用不同的原代取材方法,小胚龄可取全脑组织培养,而大胚龄则可取纹状体等特定脑区进行培养。不同胚龄或脑区来源的NPC星形胶质细胞及神经元分化比例一致。
Background and objective
Ischemic-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.
Methods
1. An effective method of non-viral vectors mediated exogenous genes transfection in the immortalized neural progenitor cells
The 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 genes
The 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 INPC
The 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 mechanism
Under 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 analysis
Data 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.
Results
1. 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.
Conclusions
1. 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.
Summary
In 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.
引文
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