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

发布时间：2018-01-19 23:12

本文关键词： NF-κB 转染凋亡存活神经前体细胞永生化基因 bcl-2 细胞培养神经前体细胞细胞分化时间特异性空间特异性　出处：《华中科技大学》2007年博士论文　论文类型：学位论文

【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,P0.05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P0.05)。 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(P0.05),并且缺氧缺糖12 h后,细胞存活率低于其它细胞株(P0.05)。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(P0.05)。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。
[Abstract]:Background and purpose of research
Hypoxic ischemic brain damage is one of the common diseases of a serious threat to human life and health, which leads to the loss of neural tissue, the traditional drug treatment method is not satisfactory. The neural stem cells or progenitor cells for tissue regeneration and repair brings hope. Neural stem cells are self-renewal ability, immature the ability of cell proliferation and differentiation potential. It is in the development of nervous system, play an important role in the maintenance and repair of normal brain function and brain injury. But the study found that due to ischemia or hypoxia microenvironment change, migration of endogenous or exogenous transplanted neural stem cells or progenitor cell survival number the survival time is short, it is difficult to play an effective role. Repair of nuclear factor kappa B (NF- K B) is a nuclear transcription factor that can regulate the expression of a series of genes. It is composed of 5 subunits, p65 And p50 is one of the most important of the two subunits of.NF- kappa B is widely expressed in the nervous system, under ischemic condition, surrounding tissue ischemia in NF- kappa B was activated, but it still play a protective role in apoptosis, remains controversial. These phenomena make us pay attention to such a problem, ischemia around the focus of NF- kappa B activation on the survival of neural stem cells or progenitor cells have no effect? The intervention of NF- kappa B can become a new strategy to improve the neural precursor cells in brain injury?
This project is intended to immortalized neural progenitor cells (INPC) cells model by NF- kappa B subunit P50, p65 gene transfection to investigate its effects on neural precursor cell survival and its mechanism, and then clear NF- kappa B on neurogenesis and neural precursor cells in hypoxic ischemic injury. For the further study of neural precursor cells to repair in treatment of ischemic brain injury and laid a theoretical basis.
research method
Selection of 1. non viral vectors mediated by exogenous gene into immortalized neural precursor cell line
Using non viral vector cationic liposome Lipofectamine 2000, TRANSfection or Sofast were loaded cationic polymer transfection of plasmid EGFP-C1 INPC, 24 h after transfection. The transfection efficiency was detected on the transfection efficiency of a vector with the highest observed after transfection, the transfection efficiency of H and 12,24,48 72, to determine the expression peak of EGFP. Using the trypan blue exclusion test activity transfected and untransfected cells. After transfection of INPC plasmid EGFP-C1, the neomycin analogue G418 screening, selection of stable cell clones of INPC/EGFP, the positive rate of EGFP was observed. The application of nestin (Nestin) antibody INPC/EGFP INPC/EGFP. induced differentiation of fetal bovine serum, cell morphology and the expression of EGFP was observed after differentiation. The expression of immune cell chemistry detection of Lipofectamine 2000 liposome mediated RcCMV-p65 plasmid was transfected to INPC after p65.
Construction of an immortalized neural precursor cell line modified by 2.NF- kappa B subunit P50, p65 gene
Using liposome Lipofectamine 2000 P50 and p65 encoding plasmid RcCMV-p50, RcCMV-p65 and Rc/CMV respectively control empty plasmid transfected immortalized rat neural progenitor cells (INPC). After G418 screening, positive clones. With the method of limited dilution separation of single cell cloning and expand culture. The selection of P50 or p65 expression in single cell clone with the highest using immunocytochemistry and Western blotting. Transient transfection of RcCMV-p50 plasmid in selected p65 expression in single cell clone.RT-PCR detection with the highest amount of neomycin (Neo) gene, the expression of P50 or p65 gene transcription. To detect the expression of P50 or p65 protein by Western blotting.
3. the formation, distribution and transcriptional activity of different two polymer after transfection of NF- kappa B subunit
The NF- kappa B subunit P50, p65 gene modified different immortalized neural progenitor cells by electrophoretic mobility shift assays (EMSA) detected in the nucleus of NF- kappa B binding activity of DNA inhibitory protein. Analysis of cytoplasmic NF- kappa B immunoblotting (I kappa B alpha) expression of P50 and p65 detection. Immunocytochemical method respectively in the cell nucleus and cytoplasm content distribution. By using the luciferase reporter assay of each cell line NF- kappa B transcription activity.
The effect of 4.NF- kappa B on the survival of immortalized neural precursor cells and its related mechanism
Glucose deprivation for 1 h or 3 h in normal and hypoxic conditions, NF- kappa B transcription activity of luciferase reporter system to detect the cell lines Annexin, V and propidium iodide (PI) to detect the cell apoptosis rate of double labeling flow cytometry. OGD 6 h, Hoechst 33342 bisbenzimide staining the nucleus, cell morphological changes were observed by PI staining, cell apoptosis rate were detected by flow cytometry. 12h after OGD, four methyl thiazolyl tetrazolium colorimetric test (MTT test) to determine the cell survival rate. 6 h OGD in normal and hypoxia, B analysis of each cell line NF- kappa wither dead the related target gene Bcl-2, Bax, Bcl-Xs/l, FAS-L, p53 blotting, the expression of XIAP and Actin protein by ImageQuant TL analysis software, Amersham band densities, calculate the ratio of Bax/Bcl-2 cells.
5. statistical analysis
SPSS11.5 statistical software was used to analyze the data, and the measurement data were expressed by mean + standard deviation (+ s). Multiple sample mean comparisons between groups were repeated measure two factor ANOVA or one-way ANOVA, and P0.05 was statistically significant.
Research results
1.Lipofectamine 2000, TRANSfection 24 h and Sofast INPC after transfection, the transfection efficiency was (25.5 + 2.9)% and (4 + 1.7)% and (7.9 + 1.4)%, Lipofectamine 2000. The highest transfection efficiency after transfection, the transfection efficiency of H 12,24,48 and 72 respectively (17.1 + 0.7)%, (25.5 + 2.9)% and (19.4 + 0.9)% and (15.6 + 1.4)%, EGFP at 24 h after transfection was the highest in.INPC/EGFP, the positive rate of EGFP is about 95%.INPC/EGFP. The expression of nestin positive cells were neurons or astrocytes, transient transfection and cell bodies and processes are still visible green fluorescence 2000.Lipofectamine mediated RcCMV-p65 plasmid, some cells showed p65 positive staining, the positive rate is about 15%.
2. by stable screening and limited dilution method isolated single cell clones stably transfected with p65 A12, A13, B11, C21, C22 and E2. p65 staining and Western blot analysis were positive, but C21 p65 was stronger than other clones. The C21 clone named INPC/p65 cell lines, and for subsequent experiments. Again the transient transfection of RcCMV-p50 plasmid, INPC/p50p65 cell lines stably transfected with RcCMV-p50. Also, the single cell clone of RcCMV plasmid, named INPC/p50 and INPC/CMV cell strain.Neo gene RT-PCR showed that control empty plasmid was successfully transfected into INPC/ CMV.RT-PCR and Western blot analysis showed that P50 and p65 genes were highly expressed in transfected with the corresponding plasmid in cell lines.
3.EMSA INPC/p50, INPC/p65 and INPC/p50p65 showed that the cell nucleus were formed homodimer with two P50, two p65 homologous dimer, p50p65 heterologous dimer and two P50 homology two dimers.INPC/p65 and INPC/p50p65 cell lines, the expression of I kappa B alpha in the cytoplasm increased. Immunocytochemistry showed that as the main nuclear P50 strong positive staining of INPC/p50 cells, but INPC/p65 cells mainly in cytoplasm of p65 positive staining, but in some cells were also found in the nuclei of the positive staining of p65.NF- kappa B transcription activity in INPC/p50 cells increased slightly in INPC/p65, significantly increased INPC/p50p65 cells in the INPC/p65 cell line the highest (P0.05).
The 4. KB B dependent luciferase activity in NF- kappa B subunit in transfected cell lines between different and different hypoxia treatment group (control group, OGD 1 h group, OGD 3 h group) have detected significant differences.Annexin V-FITC apoptosis, INPC/p65 and INPC/p50p65 cell lines occurred spontaneous cell apoptosis, and to OGD stimulation is more sensitive to hypoxia and lack of glucose. After 6 h, the cells can be observed morphological changes of apoptosis, flow cytometry confirmed that the apoptosis of INPC/p65 and INPC/p50p65 cell lines was increased (P0.05), and 12 h after OGD, cell survival rate is lower than the other cell line (P0.05) in normal and hypoxia treatment under the condition of 6 h, the ratio of expression of Bax and Bcl-2 protein and both increased in INPC/p65 and INPC/p50p65 cell lines (P0.05).
research conclusion
1.Lipofectamine 2000 can efficiently and easily transfect INPC., which lays an experimental foundation for exploring the effect of NF- kappa B gene on the biological characteristics of immortalized neural progenitor cells.
2. the immortalized rat neural precursor cells modified by NF- kappa B subunit P50 and p65 gene were successfully constructed.
The high expression of 3.p50 and p65 can escape the blocking effect of endogenous I kappa B alpha in the cytoplasm, resulting in the formation of different NF- kappa B two dimers, and directly increase the transcriptional activity of NF- kappa B.
4., the activity of NF- kappa B transcriptional activity induced by different subunits of NF- kappa B can induce spontaneous apoptosis of neural precursor cells and is more sensitive to apoptotic stimuli, which may be mediated by Bcl-2 family dependent pathway.
Research Summary
This project constructed NF- kappa B subunit P50 by liposome transfection technique, p65 gene modified immortalized neural progenitor cells, and confirmed that P50, inhibition of the expression of p65 can run in the cytoplasm of endogenous I kappa B alpha in the nucleus, leads to the formation of different NF- kappa B two dimer and, the direct increase of NF- kappa B transcription activity, and observed elevated NF- kappa B transcription activity leads to spontaneous apoptosis of neural precursor cells, and more sensitive to apoptotic stimuli, this phenomenon may be the family of Bcl-2 dependent pathway. The study will help us to define NF- kappa B in neurogenesis and hypoxic ischemic neural stem cells or progenitor cells injury, inhibition of NF- K B activity may be a new strategy to improve the neural precursor cells in brain injury.
Objective to study the human embryo of different embryonic neural precursor cells or old brain regions (NPC) in vitro differentiation and proliferation characteristics. Methods the human fetal brain tissue cells into embryonic cerebral small groups, large embryo age whole brain group, large embryonic neocortex, larger embryonic striatum group, 14-17 the age group, large embryonic midbrain group, large embryo age group and cerebral medulla Group 8 large embryo age group, suspension culture. The expression of Nestin antigen identified cells, differentiation and self-renewal ability were observed. Cell growth, proliferation. Using immunofluorescence method to compare small embryo age of whole brain group the larger, embryonic whole brain group, large embryonic neocortex, larger embryo age group and larger embryonic striatum group diencephalic differentiation of neurospheres, neurons and astrocytes. The proportion of suspension cell culture results were nestin positive antigen, can differentiate into microtubule associated Protein 2 (MAP2) or glial fibrillary acidic protein (GFAP) positive cells, and 5- bromo -2- deoxyuridine (BrdU) incorporation test positive. One week culture, the number of neurospheres larger embryonic striatum group, the morphological rules, followed by the larger embryo age diencephalon group, small embryonic cerebral group the larger, embryonic whole brain group, large embryonic neocortex group, other groups were individual neurospheres. Using limited dilution method can choose single cell clone ball from the larger embryo age group. Small embryonic striatal brain group, large embryo age whole brain group, large embryonic neocortex, larger embryonic striatum the larger embryo age group and diencephalon group NPC after induction of differentiation, MAP2 or GFAP positive cell rate difference between groups was not significant. Conclusion from the human nerve central nervous system from different embryonic age and brain precursor cells were amplified in vitro and in different embryonic age can adopt different primary sampling method. Small embryo Age can be used for whole brain tissue culture, while large embryo age can be used to culture specific brain regions, such as striatum. NPC astrocytes and neurons differentiated from different embryonic ages or brain regions are in the same proportion.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329

【参考文献】