【作者】 刘玉霞；

【导师】 汪泱；

【作者基本信息】 南昌大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 目的和意义:脑缺血及脑损伤是严重威胁人类生命健康的多发病,其主要的病理损害是神经元的损伤和缺失,导致神经功能缺损。直到上世纪90年代,神经再生与神经干细胞(neural stem cells,NSCs)的发现为中枢神经系统损伤的修复带来了希望。NSCs具有强大的增殖能力和多分化潜能,病理情况下,内、外源性的NSCs能够增殖并向病灶区迁移,最终分化成各种类型的神经元和神经胶质细胞,以修复损伤的神经系统。但研究发现内、外源性NSCs的存活、增殖能力有限,并且易分化为胶质细胞;而微环境对NSCs的增殖、分化起着决定性作用。因而,如何调控NSCs的增殖及分化方向,是应用NSCs治疗神经系统疾病之前急需解决的问题。近年来研究发现,核因子-κB(nuclear factor-kappa B,NF-κB)信号系统与中枢神经系统疾病发生发展密切相关,并且可能参与了NSCs增殖分化的调控,但其确切机制尚不清楚。本实验通过观察NF-κB信号通路激活剂(肿瘤坏死因子α,tumor necrosis factor-alpha,TNF-α)、NF-κB信号通路抑制剂(四氢化吡咯二硫代氨基甲酸盐,pyrrollidine dithiocarbamate,PDTC)对体外培养的NSCs增殖状况的影响,探讨NF-κB在NSCs增殖过程中的调控作用;同时初步探讨稀土化合物氯化镧(Lanthanum chloride,LaCl3)对体外培养的NSCs增殖状况的可能影响,以进一步了解NSCs增殖分化的调控机制,为更好地利用NSCs治疗神经系统疾病提供理论依据。方法:1、小鼠神经干细胞(mouse neural stem cells,mNSCs)的分离培养及鉴定:采用成球悬浮法分离培养mNSCs,至第三代后,行细胞冰冻切片,免疫组化方法鉴定NSCs标志蛋白nestin。2、实验分组:将生长状态良好的第3代mNSCs分为空白对照组、TNF-α组、PDTC组,分别采用常规NSCs培养液、含10ng/ml TNF-α的NSCs培养液、含0.1mmol/l PDTC的NSCs培养液培养30分钟、4小时和72小时;另设一组氯化镧组,以含氯化镧2.5μmol/L的NSCs培养液培养NSCs 1~3天。3、观察指标及方法:采用免疫荧光细胞化学方法及Western Blotting方法检测NSCs NF-κB活化状况(即P65在细胞中的分布变化);采用免疫荧光细胞化学方法检测与细胞增殖相关的指标(包括CyclinD1、Ki-67)及神经球计数、神经球体积测量方法观察NSCs的增殖状况。结果:1、mNSCs的生长状况及鉴定结果:mNSCs原代培养时,镜下见培养2天后细胞分裂增殖逐渐形成呈悬浮生长的形态规则的神经球,球周边不断有分裂增殖的细胞凸出。5~7天后球体积不断增大,球中央的细胞因营养缺乏而逐渐变黑,予以传代。免疫细胞荧光法鉴定神经球中大部分为Nestin表达阳性细胞。2、NSCs的NF-κB活化状况:细胞免疫荧光染色结果显示,TNF-α组可见明显的p65活化由胞浆转位至胞核;而PDTC组P65仍表达在胞浆,未见核转位。Western Blotting结果显示,TNF-α组胞核NF-κB/p65蛋白含量显著高于对照组,而PDTC组胞核p65蛋白含量极少。结果表明,TNF-α能使mNSCs NF-κB活化。3、NSCs的增殖状况:神经球计数和神经球体积测量结果显示,TNF-α组和氯化镧(2.5μmol/L)组神经球数量分别为[(74.56±2.6)个/瓶]和[(62.32±2.8)个/瓶],较对照组[(42.28±3.5)个/瓶]明显增多,P<0.05;神经球体积分别为[(0.82±0.16)×106(μm3)]和[(0.66±0.12)×106(μm3)],较对照组[(0.26±0.08)×106(μm3)]明显增大,P<0.05。细胞免疫荧光染色结果显示,TNF-α组和空白对照组中,CyclinD1阳性细胞率分别为(68.6±4.2)%和(38.8±3.7)%,明显高于PDTC组的(24.2±2.8)%,p<0.01;Ki-67阳性细胞率分别为(48.2±3.6)%和(35.2±4.7)%,明显高于PDTC组的(18.7±1.8)%,p<0.01。以上研究结果表明,TNF-α可使mNSCsNF-κB活化,并促进NSCs增殖;PDTC能抑制mNSCs NF-κB的活化,从而抑制NSCs增殖;一定浓度的氯化镧可促进mNSCs增殖。结论:1、NF-κB的激活或抑制可促进或抑制NSCs的增殖,NF-κB在NSCs的增殖过程中可能起着重要的调控作用。2、一定浓度的氯化镧具有促进NSCs增殖的作用。更多还原

【Abstract】 ObjectiveIschemic cerebrovascular disease and brain injury have become one of the main dangerous for the mankind health and lives. The main pathological lesions is the losses and the injury of neuron,which causes the losses of neural function.But in 1990s of last century, the discoveries of neural neogenesis and neural stem cells broght the hope for repairing the central nervous system damage. Neural stem cells have the capacity of self-renewal and multi-differentiation. In Many pathological conditions, the endogenous neural stem cells have the capacity of proliferation to migrate and to differentiate into multiple cell types such as glial cells and neurons, in order to repair the central nervous system damage.But the research find that the capacity of proliferation is limited. and easily differentiate into glial cells..The surrounding plays a crucial role in control of neural stem cell proliferation and differentiation. Thus,how to regulate neural stem cell proliferation and the direction of differentiation is the problem needs to solve for the neural stem cells applys to treat the central nervous system diseases. Recent studies find that nuclear factor-kappa B signal pathway is correlated closely with the generation and developing in the central nervous system diseases. and likely participate regulating the neural stem cell proliferation and differentiation. However, the mechanism is not clear.In the experiments, we observed that the nuclear factor-kappa B signal pathway activating stimuli such as tumor necrosis factor and the repressors such as PDTC have effect on the neural stem cells proliferative situation so as to approach the Role of nuclear factor kappa B in the proliferation of neural stem cells.In same, we explored the effects of lanthanum chloride on the neural stem cells proliferative situation so as to furtherly know the redulation mechanisms of the proliferation and differentiation of neural stem cells.And offer the experimental rationale that the transplant of neural stem cells treat central nervous system disease.Methods1.Isolation,culture and identification of mouse neural stem cells: Mouse neural stem cells were cultured by Neural stem cell suspension method to the third passage,and identified by immunofluorescence with nestin.2.Experiment group:NSCs applied to experiment were the third passage cells and were randomly divided into the control group,TNFgroup and PDTCgroup,which were cultured in common NSCs medium , in NSCs medium supplemented with 10ng/ml TNF-αand in NSCs medium supplemented with 0.1mM PDTC for thirty minuters,four hours ,seventy-two hours, respectively. Anothor Lanthanum chloride group were cultured in NSCs medium supplemented with 2.5μmol/L LaCl3.3.Observation index and method:To detect NF-κB activity situationin of neural stem cells (that is distribution change of p65)by Immunocytochemistry and Western Blotting technique. To detect the related cell proliferation index (including CyclinD1 and Ki-67) by Immunocytochemistry and observe the proliferation situation of the neural stem cells by neurosphere counting and neurosphere volume measurement.Results1.Growth situation and identification findings of mouse neural stem cells:In primary culture, the cells proliferated into the clusters and continued to increase in size, forming small, uniform neurosphere after 2 days incubation, they will detach from the surface and floated in suspension. 5 to 7 days later, neurospheres reach a diameter hindering sufficient central nutrient supply, passaging of cells is required. Immunofluorescence analysis showed that a large number of nestin positive cells resided in neurospheres.2. NF-κB activity in neural stem cells: Immunofluorescence analysis showed that TNF group remarkblely activated the translocation of NF-κB /p65 from cytoplasm to nuclear ,while PDTC group did not occurre and expressed p65 in cytoplasm. Western Blotting analysis showed that a higher expression of p65 in TNF group compared with control group, while the P65 protein content in PDTC group is mostly none.The findings shows that TNF activates NF-κB in mouse neural stem cells.3 The proliferation situation of Neural stem cell: neurosphere counting shows that TNFgroup and 2.5μmol/L Lanthanum chloride group were 74.56±2.6 piece/bot and 62.32±2.8 piece/bot ,significantly increased more than control group (42.28±3.5 piece/bot),P<0.05;and neurosphere volume measurement shows that TNFgroup and 2.5μmol/L Lanthanum chloride group were(0.82±0.16)×106(μm3)and(0.66±0.12)×10(6μm3), significantly increased larger than control group(0.26±0.08)×10(6μm3),P<0.05. Immunofluorescence analysis showed that the percentage of CyclinD1 positive cells were(68.6±4.2)% and (38.8±3.7)%in TNF group and control group were remarkblely higher than PDTC group,which was (24.2±2.8)%,p<0.01.while the percentage of ki-67 positive cells were(48.2±3.6)% and (35.2±4.7)%in TNF group and control group were remarkblely higher than PDTC group,which was (18.7±1.8)%,P<0.01。The upper results show that TNF activates NF-κB in mouse neural stem cells and significantly activated NSC proliferation,while PDTC inhibits NF-κB in mouse neural stem cells and significantly inhibited NSC proliferation 2.5μmol/L LaCl3.can promote the proliferation of mouse neural stem cells.Conclusions1. The activation or inhibition of the NF-κB can activate or inhibit neural stem cells proliferation and NF-κB is a crucial regulator of NSCs proliferation2. 2.5μmol/L Lanthanum chloride can activate neural stem cells proliferation更多还原