【作者】 王永红；

【导师】 刘宏亮；

【作者基本信息】 第三军医大学 ， 康复医学与理疗学， 2007， 硕士

【摘要】 中枢神经系统修复是康复医学的热点和难点。在成年人中枢神经系统功能损害性疾病中,脑血管疾病的比例当居首位,其次为神经系统变性疾病、颅脑和脊髓损伤等。各种中枢神经系统疾病和损伤虽病因各异,但病理上均有中枢神经系统不同部位、不同程度的神经元缺失和结构功能异常。神经干细胞(Neural stem cells,NSCs)是指中枢神经系统(Central nervous system,CNS)广泛存在的具有自我更新、自我增殖能力和多种分化潜能的特殊细胞群。神经干细胞移植治疗中枢神经系统疾病和损伤近年来受到了广泛的关注,可达到修复神经通路以及重建神经功能的目的,为临床实践带来了希望。目前认为,神经干细胞增殖分化的机制十分复杂,受到正性和负性调节分子的调控。各种调控NSCs命运的信号分子是相互协同和依赖的,NSCs所具有的内在分化程序、局部环境中的细胞因子、细胞外基质、黏附分子、细胞间的相互作用等均与其增殖、分化密切关系。细胞因子的效应又受多种因子的调节,包括SOCS(Suppressors of cytokine signaling,细胞因子信号抑制因子)家族。Id(Inhibitor of DNA Binding,DNA结合抑制因子)作为bHLH(Helix-loop-helix,碱性螺旋-环-螺旋)转录因子的负性调节子而起作用。Id2是Id家族成员之一,原位杂交显示在脑和脊髓有广泛表达,对神经系统发育有着重要的功能,通过阻滞bHLH转录因子作用,抑制NSCs向神经元分化。SOCS是近年来发现的一类可被多种细胞因子诱导产生,包括多种细胞因子、生长因子和激素,并对细胞因子信号通路具有负反馈调节作用的蛋白分子。SOCS2是SOCS家族成员之一,SOCS2 mRNA在发育和成年动物神经系统中均有表达,主要限定在神经元中表达。研究表明SOCS2是一种潜在的神经分化调控因子,在控制神经细胞分化和决定细胞命运方面起重要作用,能促进NSCs向神经元方向分化,促进神经突起生长,对神经元的结构和功能均具有重要作用。目前国内外神经干细胞的增殖及定向分化研究,多关注细胞因子对NSCs的影响。中医药是我国的“国粹”,在神经康复的实验研究和临床应用中具有巨大潜力。因此应用中药诱导NSCs的增殖和分化,促进神经再生和神经功能的修复与重建,可望为中药治疗中枢神经系统疾病和损伤开辟重要的治疗途径。但中医药对神经干细胞增殖分化的研究多局限于现象观察,缺乏深入的机制研究。银杏(Ginkgo bilobo)是一古老而神奇的植物,有“活化石”之称。银杏萜内酯为银杏叶提取物(EGb,extract of ginkgo bilobo)的主要有效成分,其中银杏内酯B(GKB,Ginkgolide B)含量最多,且抗人体血小板活化因子(PAF)作用最为强大。有研究发现,银杏叶提取物能促进周围神经再生,GKB具有促进NSCs分化的作用,但其作用机制仍不清楚,进一步阐明其调控机制对中医药在神经修复领域深层次研究开辟思路。目的:探讨银杏内酯B对神经干细胞分化的影响及其作用机制,为GKB应用于神经康复实验研究和临床应用提供一定的基础。方法:从SD大鼠胎鼠SVZa(Anterior subventricular zone,室管膜前下区)分离和培养NSCs。在加入5%胎牛血清(FBS)和不同浓度GKB的分化培养基中培养6h、12h、24h、3d和7d,用倒置相差显微镜测量分化细胞突起数目、突起长度和胞体面积,用免疫荧光细胞化学染色方法检测其微管相关蛋白(β-Tubulin)、胶质纤维酸性蛋白(GFAP)及少突胶质细胞特异性蛋白(CC-1)的表达,并计算阳性细胞的百分率。同时检测细胞因子SOCS2、转录因子Id2的表达,计算SOCS2、Id2阳性细胞百分率、SOCS2、Id2免疫反应物的平均光密度值。结果:1.NSCs培养、鉴定。从SD大鼠胎鼠SVZa成功分离和培养NSCs,传代后Nestin染色阳性鉴定。2.银杏内酯B组分化细胞的神经突起数目增多、长度增长、胞体面积增大。在相同时相点(24h或3d、7d),20mg/L、40mg/L、60mg/L GKB组神经突起数目、平均长度、胞体面积与对照组比较均有显著差异(P＜0.01);40mg/L、60mg/L GKB组神经突起数目、突起长度、胞体面积与20mg/L GKB组比较有显著差异(P＜0.01),但40mg/LGKB组与60mg/L GKB组比较无显著差异(P＞0.05)。在不同时相点组间比较(24h和3d),在突起长度方面,各GKB组和对照组3d突起长度与24h突起长度比较均有显著差异(P＜0.01);在突起数目方面,对照组3d突起数目比24h突起数目有所增加,但无显著差异(P＞0.05),而各GKB组3d突起数目比24h突起数目比较有显著差异(P＜0.01)。3.银杏内酯B组β-Tubulin阳性神经元样细胞百分率增加,GFAP阳性星形胶质样细胞百分率随GKB浓度的增加而增加,CC-1阳性少突胶质样细胞百分率无明显变化。在相同时相点(3d、7d),20mg/L、40mg/L、60mg/L GKB组β-Tubulin阳性神经元样细胞百分率较对照组明显升高(P＜0.01),40mg/L GKB组、60mg/L GKB组β-Tubulin阳性神经元样细胞百分率与20mg/L GKB组比较有显著差异(P＜0.01),但40mg/L GKB组与60mg/L GKB组比较无显著差异(P＞0.05);20mg/L、40mg/L、60mg/LGKB组GFAP阳性星形胶质样细胞百分率与对照组比较有明显升高(P＜0.01),各GKB组组间比较均有显著差异(P＜0.05-0.01)。各GKB组CC-1阳性少突胶质样细胞百分率与对照组比较无显著差异(P＞0.05)。4.银杏内酯B组转录因子Id2阳性细胞百分率及免疫反应物的平均光密度值减少。在时相点7d,对照组、20mg/L、40mg/L、60mg/L GKB组Id2阳性细胞百分率分别为66.24%、57.46%、51.52%、52.03%,Id2免疫反应物的平均光密度值分别为5.272、4.457、3.820、3.702。经统计分析,20mg/L、40mg/L、60mg/L GKB组Id2阳性细胞百分率和免疫反应物的平均光密度值较对照组显著减少(P＜0.01);40mg/L、60mg/L GKB组Id2阳性细胞百分率和免疫反应物的平均光密度值与20mg/L GKB组比较有显著差异(P＜0.01),但40mg/L GKB组与60mg/L GKB组比较无显著差异(P＞0.05)。5.银杏内酯B组细胞因子SOCS2阳性细胞百分率及免疫反应物的平均光密度值增加。在时相点7d,对照组、20mg/L、40mg/L、60mg/L GKB组SOCS2阳性细胞百分率分别为22.17%、30.24%、35.46%、34.72%,SOCS2免疫反应物的平均光密度值分别为1.134、1.382、1.578、1.527。经统计分析,20mg/L、40mg/L、60mg/L GKB组SOCS2阳性细胞百分率和免疫反应物的平均光密度值与对照组比较有显著差异(P＜0.01);40mg/L、60mg/L GKB组SOCS2阳性细胞百分率和免疫反应物的平均光密度值与20mg/L GKB组比较有显著差异(P＜0.01),但40mg/L GKB组与60mg/L GKB组比较无显著差异(P＞0.05)。结论:GKB使分化的神经细胞神经突起数目增多、长度增长、胞体面积增大;GKB提高分化细胞中神经元样细胞百分率,星形胶质样细胞百分率与银杏内酯B呈剂量依赖关系,对少突胶质样细胞百分率则无明显影响;GKB增加SOCS2阳性细胞百分率及免疫反应物的平均光密度值,减少Id2阳性细胞百分率及免疫反应物的平均光密度值。综上所述,GKB促进分化细胞神经突起生长和形态结构成熟,促进NSCs向神经元和星形胶质细胞分化,上调SOCS2表达,下调Id2表达。SOCS2、Id2可能是GKB对NSCs分化影响的作用机制中的重要环节和重要因素,GKB通过上调SOCS2表达,下调Id2表达,发挥促进神经元分化和结构功能成熟的生物学效应。这些初步研究为银杏内酯B对神经系统疾病治疗和损伤修复等实验研究和临床应用提供一定的资料和基础。更多还原

【Abstract】 Repair treatment of central nervous system is popular and difficult in the physical medicine and rehabilitation. Among the central nervous system diseases and injuries, cerebral vascular diseases have the highest proportion, followed by the nervous system degenerative diseases, brain and spinal cord injury. Though the central nervous system diseases and injuries have different etiology, but all have various degrees of neuronal loss and structure damage and dysfunction on pathology. Neural stem cells (NSCs) exist widely in the mammalian central nervous system and posses the capability of self-renewing, self-duplication, and the potentiality of differentiating into various neural cells. Neural stem cell transplantation in the treatment of central nervous system diseases and injuries in recent years attracts much attention, can be achieved to repair nerve pathways, and make nerve function recover, brings hope in the clinical practice.It is well know that the mechanism of proliferation and differentiation of NSCs is very complicated which is regulated by positive and negative regulator. Various signaling molecules which controling the fate of NSCs is synergy and mutual dependence, the inherent differentiation process of NSCs, cytokines, extracellular matrix, adhesion molecules in the local environment, the interaction between the cells all may be closely related to proliferation, differentiation of NSCs. The effect of cytokines also is regulated by many factors, including SOCS (signaling Suppressors of cytokines) family.Id (Inhibitor of DNA Binding) works as a negative regulator of bHLH (Helix-loop-helix) transcription factor. As a member of Id family, Id2 broadly expresses in the brain and spinal cord in situ hybridization and has an important effect in the development of central nervous system. Id2 can block the function of bHLH transcription factor, inhibit NSCs differentiate into neurons. SOCS (Suppressors of cytokine signaling) proteins discovered recently can be induced by a wide range of cytokines including growth factors and hormone. They work as negative feedback regulators of cytokine signal pathways. As a member of SOCS family, researchers show that SOCS2 mRNA is expressed in many tissues of embryonic and adult animals, especially in neuron. Recently studies showed that SOCS2 was a potent regulator of neural differentiation, and played an important role in the control of neural differentiation and cell fate. It can promote NSCs to differentiate into neurons, promote neurite growth. Therefore, SOCS2 plays an important role in both structure and function of neuron.Currently the study of proliferation and differentiation on NSCs concentrated on cytokines in the domestic and international. Chinese medicine is our national essence, has tremendous potential in the experimental research and clinical application. Therefore application of Chinese medicine on proliferation and differentiation of NSCs to promote nerve regeneration and restore nerve function, can opened up a new therapeutic approaches on the treatment of central nervous system diseases and injuries. But the studies of Chinese medicine on neural stem cell proliferation and differentiation limit on the phenomenon observation, and lack of in-depth study on the mechanism. Ginkgo is an ancient, magical plant, famous as "living fossil". Ginkgolides is the main active ingredient of Ginkgo biloba extract (EGb), and Ginkgolide B (GKB) is the largest effective component, and effect of anti-human platelet-activating factor (PAF) is the most powerful. Researchers found that Ginkgo biloba extract can promote regeneration of peripheral nerve, GKB can promote NSCs differentiation into neuron, but its mechanism is not yet clear. Therefore, the objective of this study was to observe the effect of GKB of various consistency on the differentiation of NSCs, explore initially the mechanisms of its action and provide a basic research about GKB in neurological experimental studies and clinical rehabilitation.To answer these questions, firstly NSCs were dissociated from SVZa of lateral cerebral ventricle of fetal SD rats. Secondly, cultured in differentiation medium containing 5% fetal bovine serum (FBS) and GKB for 6h、12h/ 24h、3d and 7d, the NSCs were induced to differentiation by different consistency of GKB to observe the effects of GKB on neurite outgrowth and the differentiation of NSCs. the neurite number, length and cell body area were photographed and measured by inverted phase-contrast micrograph, then Microtubule-associated protein(β-Tubulin)、Glial fibrillary acidic protein (GFAP)、Oligodendrocyte-specific protein (CC-1) expression were detected and counted by fluorescence microscope. Thirdly, cytokine factory-SOCS2、transcription factory -Id2 also were immunostained. The percentage of SOCS2、Id2 positive cells and the mean optical densities of immunoreactive products were obtained respectively.The main results are as follows:1. Successful isolated NSCs from anterior subventricular zone of lateral cerebral ventricle (SVZa) of fetal SD rats, cultured SVZa NSCs in vitro and identificated by Nestin-positive expression.2. Ginkgolide B increases neurites number and length and cell body area . At the same time (24h or 3d、7d), the neurite number、length and cell body area in the 20 mg / L、40 mg / L、60 mg / L GKB group were significantly increased than that in the control group (P <0.01); the neurite number、length and cell body area in the 40 mg / L、60 mg / L GKB group were significantly increased than that in 20mg/L GKB group (P <0.01), but there was not significantly different between 40 mg / L and 60 mg / L group (P> 0.05). At the different time (24h and 3d), the neurite length on 3d in the GKB groups and the control group were markerly increasd than that on 24h (P <0.01); the neurite number in the control group was not significantly different between 24h and 3d (P> 0.05), while the neurite number in GKB groups was significantly increased between 24h and 3d (P <0.01).3. Ginkgolide B increases the percentage ofβ-Tubulin positive neuron-like cells and GFAP positive astrocyte-like cells, but has no effect on CC-1 positive oligodendrocyte-like cells. The percentage ofβ-Tubulin positive neuron-like cells in GKB groups was significantly higher than that in the control group. The percentage ofβ-Tubulin positive neuron-like cells in the 40 mg / L、60 mg / L GKB group were significantly increased than that in 20mg/L GKB group (P <0.01), but there was not significantly different between 40 mg / L and 60 mg / L group (P> 0.05). The percentage of GFAP positive astrocyte-like cells in GKB groups was significantly higher than that in the control group, and there were significantly different in the GKB groups (P <0.05-0.01 ), The percentage of GFAP positive astrocyte-like ells increase in a dose-dependent manner with various consistency of GKB. But there were no significantly differences of CC-1 positive oligodendrocyte-like cells among GKB groups and the control group.4. Ginkgolide B decreases the percentage of Id2 positive cells and the mean optical densities of Id2 immunoreactive products. The percentage of Id2 positive cells in the control group、20mg/L、40 mg/L、60 mg/ L GKB group was 66.24%、57.46%、51.52%、52.03%, and the mean optical densities of Id2 immunoreactive products was 5.272、4.457、3.820、3.702. The percentage of Id2 positive cells and the mean optical densities of Id2 immunoreactive products in GKB groups were significantly lower than that in the control group (P <0.01); The percentage of Id2 positive cells and the mean optical densities of Id2 immunoreactive products in 40 mg / L、60 mg / L GKB group significantly decreased compared with 20 mg / L GKB group (P <0.01), but there was not significantly different between 40 mg / L and 60 mg GKB group (P> 0.05).5. Ginkgolide B increases the percentage of SOCS2 positive cells and the mean optical densities of SOCS2 immunoreactive products. The percentage of SOCS2 positive cells in the control group、20mg/L、40mg/L、60 mg/L GKB group was 22.17%、30.24%、35.46%、34.72%, and the mean optical densities of SOCS2 immunoreactive products was 1.134、1.382、1.578、1.527. The percentage of SOCS2 positive cells and the mean optical densities of SOCS2 immunoreactive products in GKB groups were significantly higher than that in the control group (P <0.01); The percentage of SOCS2 positive cells and the mean optical densities of SOCS2 immunoreactive products in 40 mg / L、60 mg / L GKB group significantly increased compared with 20 mg / L GKB group (P <0.01), but there was not significantly different between 40 mg / L and 60 mg GKB group (P> 0.05).In summary, Ginkgolide B promotes neurite growth, enhances the neurites number and length and cell body area, promotes NSCs to differentiation into neuron and astrocyte, but has no effect on oligodendrocyte differentiation, simultaneously upregulates SOCS2 expression and downregulates Id2 expression. Socs2 and Id2 might have an important role in regulating neuronal differentiation and maturing neuronal morph and function. Ginkgolide B promotes NSCs to differentiate into neuron and mature neural structure and function by upregulating SOCS2 expression and downregulating Id2 expression. We hope our study can provide some basic data about GKB for further illustrating the mechanism of differentiation of NSCs.更多还原