【作者】 甘丽；

【导师】 耿美玉； 刘汝高；

【作者基本信息】 中国海洋大学 ， 药物化学， 2007， 博士

【摘要】 过去相当长时间里人们认为,哺乳动物和人的脑一经发育成熟,神经细胞就不再进行增殖和分化,只有细胞的逐步退化和死亡。然而,近来大量研究表明,成体脑中存在着一类能自我增殖、分化且产生特定神经元的细胞,称之为神经干细胞。成脑的脑室和脑室下区(ventricular and subventricular zones,VZ and SVZ)及脑室周边的室管膜下区(subependymal zone),海马齿状回粒细胞下区(Subgranular Zone,SGZ)是成脑干细胞的源头。成脑干细胞的发现为干细胞治疗神经退行性疾病提供了更广阔的前景。本文利用小鼠模型,研究了人类老年痴呆(Alzheimer’s disease,AD)、帕金森氏病(Parkinson’s disease,PD)、肌萎缩侧索硬化症(Amyotrophic lateral sclerosis,ALS)三大神经退行性疾病中神经元的退行性病变、神经干/祖细胞的分布变化及神经元的再生,为AD、PD、ALS三类神经退行性疾病的发病机理提供了重要的基础资料,同时也为三类疾病的防治指明了新的方向。1. Aβ沉积对老年痴呆转基因小鼠模型中神经元再生的影响研究表明:在老年痴呆病人中,认知功能障碍与Aβ斑块的形成与大量沉积有关。然而,Aβ沉积对神经祖细胞的影响以及对神经元再生的影响仍然不是很清楚。目前已有的关于老年痴呆病人和老年痴呆动物模型中神经元再生的数据存在很大的争议。因此,我们采用了含有巢蛋白第二内含子的启动子所控制的LacZ报告基因(pNes-LacZ)的转基因小鼠(pNes-Tg)和同时含有pPDGF-APPSw,Ind和pNes-LacZ基因的双转基因小鼠(Bi-Tg)来研究Aβ沉积对脑部海马和其它区域神经祖细胞分布的影响以及对神经元再生的影响。根据体内Aβ沉积的状况,我们选择了2月、8月和12月的小鼠,分别对应于无Aβ沉积,Aβ开始沉积,Aβ大量沉积三个阶段。结果表明,与同龄对照组pNes-Tg小鼠相比,在没有Aβ沉积的2月AD双转基因小鼠中,海马区的神经祖细胞数目略有增加;在Aβ开始沉积和大量沉积的阶段,海马区的神经祖细胞数目显著减少。同时,我们发现Aβ沉积增加了海马神经元的再生但是没有明显的胶质细胞的增生。在8月,Brdu阳性细胞也显著增加。另外,Aβ斑块刚开始形成和大量沉积阶段,皮质区神经祖细胞是先增加后减少;侧脑室区,在没有Aβ沉积的2月,神经祖细胞显著增加,而当体内开始出现Aβ沉积时,神经祖细胞开始减少。Aβ沉积对体内神经祖细胞分布的影响、对神经祖细胞向神经元方向分化的影响以及对Brdu阳性细胞引起的变化揭示了刺激体内神经元的再生可能是治疗老年痴呆疾病的一个有效途径。2.锰超氧化物歧化酶对MPTP-帕金森氏症小鼠模型中多巴胺能神经元的保护作用及对神经元再生的影响PD的特征是黑质区多巴胺能神经元损伤,数目减少,导致纹状体多巴胺含量下降。目前PD病因还不是很清楚,但普遍观点认为自由基生成、氧化反应增强、谷胱甘肽含量降低以及线粒体功能异常、内源性和外源性毒物等因素,导致黑质DA神经元细胞变性,其中线粒体功能异常起主导作用。因此,我们采用了MPTP氧化损伤的PD小鼠模型(正常小鼠组)和β-actin启动子所调控的人锰超氧化物歧化酶(MnSOD)基因的转基因小鼠(MnSOD小鼠组)来研究MnSOD对多巴胺能神经元损伤的保护作用及对PD小鼠模型中神经元再生的影响。我们选取了3个时间点,即0天、10天和15天,分别代表没有注射MPTP,连续注射MPTP 10天和15天。结果表明,MPTP通过AIF和Caspase3两条凋亡通路引起正常小鼠SNC区多巴胺能神经元的凋亡,导致正常小鼠SNC区多巴胺能神经元数目的减少。尤其是在连续腹腔注射15天后正常小鼠多巴胺能神经元显著减少;而高表达MnSOD小鼠则能抑制这两条凋亡通路,神经元只有轻度减少。MPTP抑制了抗凋亡蛋白Bcl2的表达;而MnSOD小鼠维持了Bcl2的正常表达。注射MPTP后,正常小鼠组线粒体中Cyto-c、COX IV、GSH、GPX、MnSOD等抗氧化物质的表达均呈下降趋势,细胞内脂质、蛋白、DNA过氧化产物增加;MnSOD小鼠则能维持这些抗氧化物质的高表达,显著减少体内过氧化产物HNE、MDA、8-OHG、3-NT的产生。连续注射15天MPTP的正常小鼠SNC区小胶质细胞大量激活;MnSOD小鼠则没有。注射MPTP 15天时正常小鼠SNC区星形胶质细胞显著增加,Brdu免疫阳性的增殖细胞显著增加。注射了MPTP的正常小鼠和MnSOD小鼠的SNR区nestin免疫阳性的神经干/祖细胞呈增加趋势;SN区没有检测到特异性多巴胺能神经元的生成。同时,注射MPTP后,正常小鼠海马DG区nestin免疫阳性的神经干/祖细胞呈减少趋势;在10天和15天时,均可以检测到增殖的nestin免疫阳性的神经干/祖细胞。MnSOD对MPTP造成多巴胺能神经元损伤的保护作用为治疗PD药物开发提供了靶点;而MPTP损伤引起的体内神经干/祖细胞的变化及MnSOD对其的影响揭示了刺激体内神经元的再生可能是治疗帕金森氏病的一个有效途径。3.肌萎缩侧索硬化症转基因小鼠疾病发展过程中体内神经祖细胞变化的研究作用于神经干细胞或者神经祖细胞的神经再生类药物被认为是恢复ALS疾病患者损伤神经功能的途径之一。在运用再生类药物治疗ALS之前,了解体内成体神经祖细胞在运动神经元退行性病变过程中的组织与变化是极为重要的。因此,我们采用了含有巢蛋白第二内含子的启动子所控制的LacZ报告基因(pNes-LacZ)的转基因小鼠(pNes-Tg)和同时含有G93A-SOD1和pNes-LacZ基因的双转基因小鼠(Bi-Tg)来研究脊髓和脑内神经祖细胞在运动神经元退行性病变过程中的变化。结果显示:与同龄对照组pNes-Tg小鼠相比,在ALS双转小鼠疾病起始和发展阶段,脊髓背角区的神经祖细胞数目增加;而脊髓腹角区的神经祖细胞数目仅在疾病发展阶段检测到增加。在疾病开始和发展阶段,与对照组小鼠相比,在脊髓腰椎段神经祖细胞的数目远高于脊髓颈椎段和胸椎段的。在疾病整个发展过程中,与疾病起始状态相比,背角的神经祖细胞数目是降低的,而腹角的神经祖细胞数目是增加的。另一方面,运动皮质区的神经祖细胞数目在疾病起始阶段有增加,但到了疾病发展阶段,这种增加的趋势不是很明显。在侧脑室区,疾病进展期神经祖细胞的数目显著减少;而在海马区,无论在疾病的起始或进展阶段,都没有检测到神经祖细胞的变化。在ALS转基因小鼠模型中,体内神经祖细胞在疾病起始和进展阶段的重新组织与分布给刺激体内神经再生治疗ALS提供了坚实的理论基础。更多还原

【Abstract】 Last quite a long time, it was thought that once maturation, nerve cells in mammalian and human brain were no longer undergoing proliferation and differentiation, and only went to gradual degradation and death. However, a large number of recent studies show that neural stem cells also are found in adult brain, which can self-proliferate and differentiate to specific neuronal cells. Adult neural stem cells exist in brain ventricle and the subventricular zone (VZ and SVZ), ependymal and periventricular zone (subependymal zone), subgranular Zone (SGZ) in hippocampus dentate gyrus region. The discovery of stem cells in adult brain provides a broad prospect for treatment of neurodegenerative diseases with stem cell. In our experiments, mouse models of human neurodegenerative diseases for Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic Lateral Sclerosis (ALS) have been used to study neuron degeneration, distribution of neural stem/progenitor cells under the pathologic condition and neuroregeneration, which will be benefit to treatment of neurodegenerative diseases using neurogenesis in vivo and development of drugs.1. Neurogenic Responses to Amyloid-Beta Plaques in Alzheimer’s Disease-Like MiceFormation and accumulation of beta-amyloid (Aβ) plaques are associated with declined memory and other neurocognitive function in AD patients. However, the effects of Aβplaques on neural progenitor cells (NPCs) and neurogenesis from NPCs remain largely unknown. The existing data on neurogenesis in AD patients and AD-like animal models remain controversial. For this reason, we utilized the nestin second-intron enhancer controlled LacZ reporter (pNes-LacZ) transgenic mice (pNes-Tg) and Bi-transgenic mice (Bi-Tg) containing both pPDGF-APPSw,Ind and pNes-LacZ transgenes to investigate the effects of Aβplaques on neurogenesis in the hippocampus and other brain regions of the AD–like mice. We chose transgenic mice at 2, 8 and 12 months of age, corresponding to the stages of Aβplaque free, plaque onset and plaque progression to analyze the effects of Aβplaques on the distribution and de novo neurogenesis of (from) NPCs. We demonstrated a slight increase in the number of NPCs in the hippocampal regions at the Aβplaque free stage, while a significant decrease in the number of NPCs at Aβplaque onset and progression stages. On the other hand, we showed that Aβplaques increase neurogenesis, but not gliogenesis from post-mitotic NPCs in the hippocampus of Bi-Tg mice compared with age-matched control pNes-Tg mice. In addition, the number of NPCs in cortex region increased first and then decreased from Aβplaque onset to progression stages. The number of NPCs in lateral ventricle increased at Aβfree stage, and decreased when Aβplaque began to form. The alteration of NPCs distribution and neurogenic responses of NPCs to Aβplaques suggest that experimental approaches to promote de novo neurogenesis may improve neurocognitive function and provide an effective therapy for AD.2. Protective Effect of MnSOD on Depletion and Effect on Regeneration of Dopaminergic Neurons in MPTP-PD Mice ModelPD is characterized by substantia nigra pars compacta (SNC) dopaminergic neurons injury, the number reduced, causing the dopamine content. PD etiology is not clear at present, but common perception is that the production of free-radical oxidation reaction and mitochondrial dysfunction and reduced glutathione levels, endogenous and exogenous factors such as toxicants lead to degeneration of the substantia nigra dopaminergic neurons. Mitochondrial dysfunction may play a leading role. Therefore, We used MPTP- PD mouse model of oxidative damage (normal mice), and transgenic mice ofβ-actin promoter driven human manganese superoxide dismutase (MnSOD) gene (MnSOD mice) to study the protective effect of MnSOD on depletion and effect on regeneration of dopaminergic neurons in SNC of MPTP-PD mice. We used three time points, namely D0 days, D10 days, D15 days, respectively on behalf of no MPTP, MPTP with consecutive injection for 10 and 15 days. The results showed that by two apoptosis pathway of AIF and Caspase3 dopaminergic neurons were undergoing apoptosis induced by MPTP, which led to the reduction in the number of dopaminergic neurons in SNC of normal mice. Especially after intraperitoneal injection of MPTP for 15days, dopaminergic neurons in normal mice markedly reduced; while overexpression of MnSOD inhibited these two apoptotic pathways and dopaminergic neurons only slightly reduced. MPTP inhibited the anti-apoptotic protein Bcl-2 expression, and MnSOD mice maintained Bcl2 expression. With MPTP treatment, expression of antioxidant substances such as cytochrome c, cytochrome c oxidase (COX IV), glutathione (GSH), glutathione peroxidase (GPX), manganese superoxide dismutase (MnSOD) in mitochondria of normal mice tended to decrease, and peroxidative products of lipid, protein, DNA increased dramatically. MnSOD mice were able to maintain high expression of these antioxidant substances, and significantly reduced peroxidative products in vivo like 4-hydroxy-2-nonenol (HNE), malondialdehyde (MDA), 8-hydroxy-2’-deoxyguanosine (8-OHG), 3-nitro tyrosine (3-NT). With injection of MPTP for 15 days, microglia cells in SNC of normal mice were activated in a large of amount. Astrocytes and Brdu immunoreactive cells in SNC of normal mice also presented significant increases. Furthermore, with injection of MPTP, nestin immunopositive neural stem / progenitor cells in SNR of both normal mice and MnSOD mice were increased. No regeneration of dopaminergic neurons was detected in SN. At the same time, nestin immunopositive neural stem / progenitor cells in DG of normal mouse hippocampus tended to decrease. At the D10 and D15, proliferate neural stem / progenitor cells represented by nestin staining can be detected. The protective effect of MnSOD on dopaminergic neuron injury caused by MPTP provides a basis for anti-PD drug design. And the alteration of NPCs distribution and proliferation of NPCs to dopaminergic neuron injury suggest that experimental approaches to promote de novo neurogenesis may improve motor function and provide an effective therapy for PD.3. Temporal Response of Neural Progenitor Cells to Disease Onset and Progression in ALS-Like Transgenic MiceRegenerative medicine through neural stem or neural progenitor cells (NPCs) has been proposed as an alterative avenue to restore neurological dysfunction in ALS. It is critical to understand the organization and distribution of endogenous adult NPCs in response to motor neuron degeneration before regenerative medicine can be applied for ALS therapy. For this reason, we analyzed the temporal response of NPCs to motor neuron degeneration in the spinal cord and brain using nestin enhancer driven LacZ reporter transgenic mice (pNes-Tg mice: control) and Bi-transgenic mice containing both nestin enhancer driven LacZ reporter gene and mutant G93A-SOD1 gene (Bi-Tg mice). We observed an increase of NPCs in the dorsal horns of the spinal cord at the disease onset and progression stages in the Bi-Tg mice compared with that of age-matched pNes-Tg control mice. In contrast, an increase of NPCs in the ventral horns was detected at the disease progression stage. Compared to control mice, the number of NPCs in lumbar region was much higher than those of cervical region and thoracic region.On the other hand, an increase of NPCs in the motor cortex at the disease onset stage, but not at the disease progression stage was detected. Furthermore, a decrease of NPCs in the lateral ventricle at the disease progression stage was observed, while no difference in the number of NPCs in the hippocampus was detected at the disease onset and progression stages. The organization and distribution of endogenous adult NPCs in the ALS-like transgenic mice at the disease onset and progression stages provide fundamental bases for consideration of regenerative therapy of ALS by increasing de novo neurogenesis.更多还原