【作者】 肖茜；

【导师】 王宗仁； 李军昌；

【作者基本信息】 第四军医大学 ， 中西医结合临床， 2011， 博士

【摘要】 阿尔茨海默病是一种极具挑战性的神经退行性疾病,其发病机制和海马神经元有着密切的关系,它具有广泛性和复杂性的特点,呈现出较高的发病率,目前全球多达数百万的人患有此病,阿尔茨海默病已经在全世界范围内呈流行性的趋势,它的基本病理组织学特征就是在大脑内形成以β-淀粉样蛋白(beta-amyloid peptide,Aβ)为核心的神经斑,因此用Aβ25-35诱发培养海马神经细胞损伤是一种较好的阿尔茨海默病模型。银杏内酯B(Ginkgobalide B,GKB)来源于天然植物,是中药银杏叶提取物(Extract of Ginkgo bilobaleaves,EGB)的主要活性成分之一,它是一种安全、有效、经济的天然药物。现代研究表明,GKB在很多疾病方面都显示了良好的临床疗效,然而其作用机制尚不明确,所以本研究旨在观察GKB对Aβ25-35诱导的胎鼠海马神经元损伤的神经保护作用,并进一步探讨GKB可能的神经保护作用机制。我们从海马神经细胞的各种形态学改变,细胞存活率,乳酸脱氢酶的释放量,半胱氨酸蛋白酶–3(Caspase-3)的活性及细胞外钾离子的含量等变化观察GKB对Aβ25-35诱导海马神经元损伤的影响,并采用反转录-聚合酶链反应和蛋白质免疫印迹法检测脑源性神经营养因子和神经生长因子在基因水平和蛋白水平的表达变化。研究发现,GKB可以对抗Aβ25-35诱导的海马神经元的凋亡,并上调脑源性神经营养因子和神经生长因子的基因和蛋白的表达水平,改善神经再生的微环境。因此,我们认为GKB可以抑制Aβ25-35诱导的海马神经细胞损伤,而且这种抑制损伤,保护神经的作用机制可能与上调了脑源性神经营养因子和神经生长因子的表达水平,改善了神经再生的微环境有一定的相关性。本研究从多角度,多层面揭示了GKB的神经保护作用及其作用机制,为GKB用于治疗阿尔茨海默病等神经退行性疾病,促进神经损伤修复的研究奠定基础,并为天然药物的研究、开发和利用提供新的思路。第一部分:银杏内酯B的神经保护作用研究研究目的:银杏内酯B对Aβ25-35诱导海马神经元损伤的神经保护作用研究方法:取16-18 d孕龄的SD胎鼠,分离并进行海马神经元的原代培养,分别采用MTT比色法和活细胞形态学采图筛选出银杏内酯B的最佳效应时间和最佳作用浓度。海马神经元原代培养3 d后,在培养液中加入25μg / mL的Aβ25-35,4 h后再按照分组分别给药。采用已配制好在37℃下聚合7 d的Aβ25-35建立海马神经细胞损伤的模型。各组药物分别作用48 h后,在显微镜下取随机视野观察海马神经细胞的生长和凋亡情况同时进行细胞采图,并采用MTT比色法,在酶标仪下测定每孔的光密度值来观察细胞活力的大小;为了进一步研究Aβ25- 35对海马神经元的细胞毒性,我们对细胞外乳酸脱氢酶释放的含量进行检测,乳酸脱氢酶活性测定方法按照乳酸脱氢酶试剂盒说明书进行;为了研究Aβ25-35对海马神经细胞凋亡程度的影响采用Hoechst33342和PI的荧光染色法对海马细胞核进行染色,通过观察海马神经元细胞核的形态变化和荧光亮度,以检测海马神经细胞的凋亡程度;同时我们还采用NSE和Tunel荧光染色法对细胞体和细胞核共同染色,以检测Aβ25-35对海马神经细胞凋亡的影响;接着检测了细胞凋亡中的关键蛋白酶Caspase-3的活性,并检测了细胞凋亡过程中的重要离子——细胞外钾离子的含量。所有的统计学分析均采用SPSS10.0软件包,实验结果以均数±标准差( x±s)表示,实验数据采用方差分析和Fisher’s PLSD检验,以P≤0.05为差异有显著性统计学意义。研究结果:通过检测细胞活力和活细胞形态学采图,观察GKB对正常海马神经细胞的影响,我们发现与空白对照组相比较,GKB组细胞生长状态较好且光密度值明显增高(P < 0.05),表明GKB有明显的促进神经细胞生长的作用;在银杏内酯B的神经保护作用研究中,细胞形态学实验分别研究了胞体、胞核,以及胞体和胞核共同染色情况下的各种形态学指标。首先是活细胞形态学采图,当给予Aβ25-35后,Aβ25-35组细胞较空白对照组海马神经元胞体消失,产生了大量的细胞碎片,神经元突触消失,网状结构消失;而GKB+Aβ25-35组海马神经元胞体可见,细胞碎片较Aβ25-35组显著减少,海马神经元的特殊折光性可见,并且可以看到突触相互连接而成的网状结构;其次分别进行了Hoechst 33342、PI共同染色及NSE、Tunel共同免疫荧光染色,实验结果表明:当给予Aβ25-35后,凋亡细胞核数量明显增加,存活细胞核数量明显减少,胞体状态显著下降,而GKB+Aβ25-35组较Aβ25-35组凋亡细胞核数量显著减少,存活细胞核数量显著增加,细胞体的状态显著好转;细胞活力检测,乳酸脱氢酶测定,Caspase-3活性和细胞外钾离子含量的检测均表明GKB对损伤情况下的海马神经元有显著的抗Aβ25-35诱导的凋亡作用。Aβ25-35组较空白对照组乳酸脱氢酶含量、Caspase-3活性和细胞外钾离子含量均明显增加(P < 0.01),这说明海马神经细胞的神经损伤模型造模成功;而GKB+Aβ25-35组较Aβ25-35组乳酸脱氢酶含量、Caspase-3活性和细胞外钾离子含量均显著下降(P < 0.01);当加入拮抗剂后,乳酸脱氢酶含量、Caspase-3活性和细胞外钾离子含量均介于GKB+Aβ25-35组和Aβ25-35组之间(P < 0.05),说明GKB具有抗Aβ25-35诱导海马神经元凋亡的作用;GKB+Aβ25-35组和EGB+Aβ25-35组差异不明显(P﹥0.05),说明GKB和EGB有着相似的神经保护作用,并且二者都具有抗Aβ25-35诱导海马神经细胞凋亡的作用。研究结论:1.银杏内酯B对正常情况下的海马神经元有明显的促进神经元生长的作用。2.银杏内酯B对损伤情况下的海马神经元有显著的抗Aβ25-35诱导的凋亡作用。第二部分:银杏内酯B的神经保护作用机制研究研究目的:探讨银杏内酯B对Aβ25-35诱导的胎鼠海马神经元损伤的神经保护作用机制。研究方法:原代培养海马神经元,采用已配制好(在37℃下聚合7 d)的Aβ25-35(25μg / mL)建立海马神经细胞损伤的模型。实验分组如下:A:空白对照Control组,B:Aβ25-35组,C:GKB+Aβ25-35组,D:EGB+Aβ25-35组,E:GKB组,F:EGB组;分别采用Western-blot和RT-PCR的方法检测脑源性神经营养因子和神经生长因子基因和蛋白表达的变化。统计学分析采用SPSS10.0软件包,实验结果以均数±标准差( x±s)表示,实验数据采用方差分析和Fisher’s PLSD检验,以P≤0.05为差异有显著性统计学意义。研究结果:从GKB对Aβ25-35诱导的海马神经元脑源性神经营养因子与神经生长因子表达的Western-blot和RT-PCR的检测分析图中均可以看出,Aβ25-35组较空白对照组脑源性神经营养因子和神经生长因子的表达明显降低(P < 0.01),这说明海马神经细胞有明显损伤;而GKB+Aβ25-35组较Aβ25-35组脑源性神经营养因子和神经生长因子的表达显著提高(P < 0.05),这说明GKB具有一定的神经保护作用;GKB组与EGB组比较,GKB+Aβ25-35组与EGB+Aβ25-35组比较差异均不明显(P﹥0.05),这说明GKB与EGB有着相似的神经保护作用机制,且二者都可以上调脑源性神经营养因子和神经生长因子的表达水平;同时与空白对照组相比较,GKB与EGB对海马神经元均没有细胞毒性作用,且二者都可以促进胎鼠海马神经元的生长;GKB可以抑制Aβ25-35诱导的海马神经细胞的凋亡,这种神经保护作用机制是通过上调脑源性神经营养因子和神经生长因子的表达水平,从而改善了神经再生的微环境来实现的。研究结论:1.银杏内酯B和银杏叶提取物有着相似的神经保护作用机制,二者都可以上调脑源性神经营养因子和神经生长因子的表达。2.银杏内酯B对损伤情况下的海马神经元有显著的抗Aβ25-35诱导的凋亡作用。银杏内酯B的这种神经保护作用机制是通过上调脑源性神经营养因子和神经生长因子基因和蛋白的表达水平,调节了神经再生的微环境来实现的。3.银杏内酯B是中国古树银杏叶提取物中的单体成分,这种天然药物来源广泛,性质稳定,值得进一步研究、开发和利用。更多还原

【Abstract】 Alzheimer’s disease is the most challenging neurodegenerative disorder characterized by slowly progressive dementia and brain atrophy. It is a complex genetic disease in the global prevalence of up to 15 million people, and has close relationship with hippocampal neurons. Ginkgolide B is a well-defined plant extract which is safe in nature, effective and economic, and Ginkgolide B is a major component of Extract of Ginkgo bilobaleaves medicinal herb. However, the mechanisms underlying Ginkgolide B remain unclear especially in“Micro-environment for nerve regeneration”. So we investigated the potential effectiveness of Ginkgolide B against toxicity induced by Aβ25-35 on hippocampal primary cultured cells, and further exploring the possible mechanisms concerned. We first conducted the study of the Aβ25-35-induced apoptosis characterized by the changes in morphology, cell viability, lactate dehydrogenase level, the caspase-3 activity and extracellular K+ concentration in hippocampal neurons. Moreover, the expression of Brain-derived neurotrophic factor and Nerve growth factor mRNA and the protein synthesis in neurons were detected via RT-PCR and Western-blot assay. It was found out that Aβ25-35 induced apoptosis was attenuated by Ginkgolide B. Ginkgolide B caused Brain-derived neurotrophic factor and Nerve growth factor up-regulation when cells were subjected to Aβ25-35 insults. The micro-environment for nerve regeneration also improved. Ginkgolide B attenuate Aβ25-35 induced apoptosis in hippocampal neurons by Brain-derived neurotrophic factor and Nerve growth factor pattern which play a crucial role in“Micro-environment for nerve regeneration”. We consider it one of the possible mechanisms by which Ginkgolide B daunts the hippocampal neurons apoptosis.The study indicate that Ginkgolide B may significantly dampen Aβ25-35 induced apoptosis, and the neuroprotective effects may be intimately associated with Brain-derived neurotrophic factor and Nerve growth factor up-regulation caused by Ginkgolide B. These findings may demonstrate the neuroprotective effects of Ginkgolide B and offer new evidences to the possible mechanisms. The present study on the neuroprotective effects of Ginkgolide B against Aβ25-35 induced apoptosis might pave a way for further investigation of molecular mechanism underlying from protein and gene level, and also lay a foundation for clinical application in the viable molecule to treat neural degeneration diseases and nerve injury. The objective of our experiment is to study the neuroprotective effects of Ginkgolide B with a view to healing neurodegenerative disorders such as Alzheimer’s disease and advancing natural medicine research, development and utilization. Objective:Investigate neuroprotective effect of Ginkgolide B protects hippocampal neurons from apoptosis induced by beta-amyloid 25-35.Methods:Primary cultures from hippocampus of pregnant 16 to 18-day Sprague Dawley rat embryos. MTT assay and living cells morphology were used to selected the best effect time and the optimal concentration of Ginkgolide B. After 3 days in vitro, these cells were put away for further experiments. Three days after planting, Ginkgolide B was added to the culture and maintained for 4 hours before being treated with 25μM Aβ25-35. Meanwhile, positive control group, the standardized Extract of Ginkgo bilobaleaves EGb761 was added to the culture, and maintained for 4 hours before being treated with 25μM Aβ25-35. Aβ25-35 was cultured in PBS for 7 days at 37℃. After the culture was treated with different treatments, cell viability was assessed through a modified MTT assay. Since the loss of MTT is not necessarily a valid indicator of cell lysis, toxicity of Aβ25-35 was further examined through an LDH-release assay. LDH activity was determined according to the protocol of an LDH kit. To investigate if Ginkgolide B can protects hippocampal neurons against Aβ25-35-induced neurotoxicity via ameliorating cell apoptosis, we did Hoechst 33342 and PI double staining experiments. In order to identify apoptotic cells, nuclei were made fluorescent by incubation with the DNA intercalating dye. NSE and Tunel staining was carried out according to the introduction of tunel staining kit. Caspase-3 activity was measured via caspase assay kit according to the manufacturer’s instructions. To assess the permeability of neurons membranes being treated with Aβ25-35 in the presence or absence of Ginkgolide B, we detection of level of K+ leakage. Statistical analysis was made by using the computer software SPSS 10.0. The data was analyzed by using a two or one-way analysis of variance (ANOVA), then by Fisher’s PLSD post-hoc test multiple comparisons. The data was expressed as mean±S.E.M. Statistical significance was set at P≤0.05.Results:Living cells morphology: When Ginkgolide B was added, the apoptosis of the neurons caused by Aβ25-35 were improved obviously. The amount of cell debris decreased, refractivity in their bodies was promoted, and networks were formed by those neurite linking with each other. Hoechst 33342 and PI double staining: When Ginkgolide B was added, the apoptosis of the neurons caused by Aβ25-35 were improved obviously. The number of apoptotic nuclei decreases significantly, the number of surviving nuclei increase significantly. NSE and Tunel double staining: When Ginkgolide B was added, the apoptosis of the neurons caused by Aβ25-35 were improved obviously. The number of apoptotic nuclei decreased significantly, the state of cell bodies improved significantly. Effects of Ginkgolide B on cell viability, LDH release, Caspase-3 activity and Kalium ion concentration of embryonic hippocampal neurons treated with Ginkgolide B (GKB, 40μg / mL) and / or Aβ25-35 (25μM) for 48 h. The concentration of Extract of Ginkgo bilobaleaves (EGB) is 150μg / mL. The comparison on cell viability, LDH release, Caspase-3 activity and Kalium ion concentration of embryonic hippocampal neurons indicates that Ginkgolide B can protect hippocampal nerves, and Ginkgolide B has neuroprotective effect of hippocampal neurons from apoptosis induced by beta-amyloid 25-35. These figure show control group vs. Aβ25-35 group, P < 0.01; Aβ25-35 group vs. Ginkgolide B+Aβ25-35 group, P < 0.01; Aβ25-35 group vs. Ginkgolide B+I+Aβ25-35 group, P < 0.05; Ginkgolide B+Aβ25-35 group vs. Ginkgolide B+I+Aβ25-35 group, P < 0.05; Ginkgolide B+Aβ25-35 group vs. EGB+Aβ25-35 group, P > 0.05; Ginkgolide B group vs. EGB group, P > 0.05, it shows that Ginkgolide B and EGB have a similar capability of neuroprotection. Ginkgolide B and EGB both have neuroprotective effect of hippocampal neurons from apoptosis induced by Aβ25-35.Conclusions:1. Ginkgolide B has a clear role in promoting neuronal growth on normal hippocampal neurons.2. Ginkgolide B has neuroprotective effect of hippocampal neurons from apoptosis induced by beta-amyloid 25-35.PartⅡ: The mechanism of neuroprotective effect on Ginkgolide BObjective:Investigate neuroprotective mechanism of Ginkgolide B protects fetal hippocampal neurons apoptosis induced by beta-amyloid 25-35.Methods:Primary cultures hippocampus neurons. Aβ25-35 was cultured in PBS for 7 days at 37℃. After the culture was treated with the below different treatments: (A): control group; (B): Aβ25-35 group; (C): Ginkgolide B+Aβ25-35 group; (D): EGB+Aβ25-35 group; (E): Ginkgolide B group; (F): EGB group. Respectively use Western-blot and RT-PCR methods to detect changes in the expression of BDNF and NGF. Statistical analysis was made by using the computer software SPSS 10.0. The data was analyzed by using a two or one-way analysis of variance (ANOVA), then by Fisher’s PLSD post-hoc test multiple comparisons. The data was expressed as mean±S.E.M. Statistical significance was set at P≤0.05.Results:Western-blot and RT-PCR analysis of hippocampal neurons cells revealed that Ginkgolide B and Extract of Ginkgo bilobaleaves have a similar capability of neuroprotection mechanism, and both can be achieved by the up-regulation of BDNF and NGF expression. Meanwhile, it seemed that the cytotoxicity effect could not be found in Ginkgolide B group and Extract of Ginkgo bilobaleaves group, but Ginkgolide B and Extract of Ginkgo bilobaleaves could promote neuronal growth in cultured embryonic rat hippocampal neurons, compared to the control group. Furthermore, the same changes of BDNF protein and NGF protein could be detected in different groups of hippocampal neurons cells by Western-blot. Thus, BDNF and NGF were inhibited in damaged hippocampal neurons cells and the effects could be partly recovered by Ginkgolide B. The micro-environment for nerve regeneration also improved. The figure show control group vs. Aβ25-35 group, P < 0.01; Aβ25-35 group vs. Ginkgolide B+Aβ25-35 group, P < 0.01; Ginkgolide B+Aβ25-35 group vs. EGB+Aβ25-35 group, P > 0.05; Ginkgolide B group vs. EGB group, P > 0.05. The data indicates that Ginkgolide B and Extract of Ginkgo bilobaleaves have a similar capability of neuroprotection mechanism. Conclusions:1. Ginkgolide B and Extract of Ginkgo bilobaleaves have a similar capability of neuroprotection mechanism, and both can be partly achieved by up-regulation the expression of BDNF and NGF.2. Ginkgolide B has neuroprotective effect of hippocampal neurons from apoptosis induced by beta-amyloid 25-35. The neuroprotective mechanism of Ginkgolide B is via up-regulation the expression of BDNF and NGF, improve the micro-environment for nerve regeneration.3. Ginkgolide B is the extract of ginkgo bilobaleaves, the natural medicine has wide variety of sources, stable and easy to use, it is worth further research, development and utilization.更多还原