【作者】 俞英欣；

【导师】 黄远桂； 李新民；

【作者基本信息】 第四军医大学 ， 神经病学， 2009， 博士

【摘要】 Alzheimer病（老年性痴呆，Alzheimer’s disease,AD）是老年期痴呆最常见的类型，该疾病是一种进展性的神经变性病，其结果是脑功能的异常及最终的神经元凋亡。主要病理变化为由β-淀粉样蛋白（beta amyliod protein，Aβ）形成的老年斑，Aβ水平增高或者Aβ斑可能是AD神经变性的最主要原因，同时Aβ斑在AD鼠模型上也损害了海马的长时程增强。海马是贯穿整个生命阶段神经发生的主要部位，而该部位也是AD的主要影响结构。因此靶向于海马神经发生可能是治疗或者延缓AD病情的一种潜在的主要方法。然而，因为AD病人及正常人对照的脑组织标本取得是相当困难的，所以很难了解AD病人在疾病进展过程中的神经发生隋况。此研究正是为了了解随着AD病情的进展神经发生的情况，以有助于AD的治疗。携带APP及PS1突变基因的AD转基因鼠模型表现了AD的主要病理变化，并出现记忆力缺失，该模型对于AD的研究及探索AD可能的治疗方法是非常有用的。在此研究中，我们运用APP/PS1双转基因AD鼠模型来研究AD进展过程中神经发生的情况。应用水迷宫试验研究了3和9月龄转基因鼠在AD疾病进展过程中的记忆力受损情况，并与非转基因鼠进行比较，并通过Aβ染色确定AD的病理进展状况。海马神经细胞增殖是分别应用BrdU和神经前体细胞标记物doublecortin标记的方法。研究增殖细胞的分化情况，采用9月龄转基因及对照鼠BrdU注射后28天取脑，BrdU+NeuN（神经元的标记物）或者BrdU+GFAP（胶质细胞的标记物）的方法。结果如下：1．空间记忆获取是通过四个方向找到隐藏平台的时间来评定的，空间记忆保留是通过寻找目的平台周围四分之一区域的时间比例来评估的。9月龄转基因鼠水迷宫试验中出现明显的记忆力受损。2．海马Aβ斑的数量被用来确定AD转基因鼠不同月龄的病变情况。9月龄海马Aβ斑的数量显著高于3月龄转基因鼠，在非转基因对照鼠脑片上未发现老年斑形成。3．海马新生细胞由BrdU标记。9月龄转基因鼠海马区BrdU阳性细胞数（包括DG，CA1，CA3区）与对照鼠相比显著增加；而3月龄转基因鼠与对照鼠却无显著差别。4．9月龄实验鼠海马区细胞增殖同时应用DCX进行标记。转基因鼠与对照组相比海马区DG，CA1，CA3区DCX阳性细胞数显著增加。5．为了鉴定海马区新生细胞的表型，9月龄鼠BrdU注射后28天进行实验。海马DG，CA1，CA3区新生细胞的表型包括神经元（BrdU+NeuN）和胶质细胞（BrdU+GFAP）。BrdU阳性细胞表达NeuN和GFAP的比例转基因鼠相似于非转基因鼠。海马区BrdU阳性细胞的数量和分化为神经元的数量转基因鼠较对照鼠明显增加总的来说，此研究结果与在AD病人脑尸检中发现的神经发生在DG区及CA1区增加是相一致的。我们的研究是首次在APP_K670N/M671N/PS1_M146L双转基因AD鼠模型上研究神经发生。因为在该转基因鼠模型上神经发生的增加与AD病人脑部变化的相符性，所以认为该模型具有代表AD病理变化的更典型性。尽管转基因鼠海马区在生理及病理情况下神经发生的功能尚不清楚，然而研究发现成年人海马的新生细胞已经显示出某些功能。某些类型的记忆依赖于整个生命过程中新生细胞的产生。因此AD转基因鼠的神经发生可能是对病理及行为损伤的一种代偿作用。然而，尽管神经发生在AD鼠模型上是增加的，但是病理及记忆力损害却逐渐加重。可能是神经发生不足以代偿AD脑部的进展性的病理变化，或者是新生神经元尚未整合至神经网络，或者这些神经元尚无正常神经元功能。针对AD的神经发生研究需要我们继续努力，升高AD脑部的神经发生可能是AD治疗的潜在方法。更多还原

【Abstract】 Alzheimer’s disease （AD）, the most common adult onset dementia, is aprogressive neurodegenerative disease associated with dysfunction and eventualdeath of brain neurons （Price, 1986）. A pathologic hallmark of AD is theformation of senile plaques, in whichβ-amyloid peptide （Aβ） is a majorcomponent. Increased brain Aβlevels and/or Aβplaques may be the primaryinfluence resulting in neuronal degeneration in AD, and impaired long termpotentiation （LTP） in the hippocampus of AD model mice. The hippocampus isunique in continuing to produce new neurons throughout life in the structuresaffected by AD. Targeting neuronal regeneration （neurogenesis） in thehippocampus may be a potential therapy to delay or reverse the progression ofAD. However, because brain tissues from age-matched normal persons and ADpatients are generally not available, little is known about changes in endogenoushippocampal neurogenesis in the progression stage of AD. This study examinesthe changes in neurogenesis during the progression of AD with the goal ofidentifying a potential target for AD treatment. Transgenic mouse models of AD that carry an amyloid precursor protein（APP） and/or presenilinl （PS1） mutated gene show AD-like pathology andmemory impairment, and are useful for studying AD and testing possibletreatments. In this study, we used an APP/PS1 double transgenic mouse model ofAD to investigate changes in hippocampal neurogenesis through different stagesof AD. We tested the spatial memory of 3-and 9-month-old mice in a water mazeto determine the different stages of AD in the transgenic mice; the stage of ADwas confirmed by hippocampal Aβplaque staining. Hippocampal proliferatingcells in the mice were labeled by bromodeoxyuridine （BrdU） and doublecortin asmarkers for neuronal progenitor cells. To determine the differentiation ofproliferating cells, brain sections from mice injected with BrdU at 9 months andsacrificed at 10 months were double immunofluorescent labeled with antibodies,either BrdU + NeuN （a neuronal marker） or BrdU + glial fibrillary acidic protein（GFAP, an astroglia marker）.Results as followed:1. Spatial memory acquisition of the mice was assessed by escape latency in thehidden-platform test in four blocks of training trials. Spatial memory retentionwas assessed in the probe test by the time spent （percentage） searching for thetarget quadrant. Memory was impaired in the 9-month-old transgenic mice in awater maze test.2. The number of hippocampal Aβplaques was used to confirm the differentstages of AD amyloid pathology in the different aged transgenic mice. Amyloidpathology was increased in the 9-month-old transgenic mice. The number ofhippocampal Aβplaques in the 9-month-old transgenic mice was significantlyhigher than in the 3-month old transgenic mice. No plaques were detected in thenontransgenic mice. 3. Cell proliferation in hippocampus of the mice was labeled by BrdU staining.In the 9-month-old mice, the number of BrdU-positive cells in the hippocampus,including dentate gyms （DG）, CA1, and CA3 was significantly higher in thetransgenic mice than in the nontransgenic mice; these differences were notobserved in the 3-month-old mice.4. Cell proliferation in the hippocampus of 9-month-old mice was also labeledby DCX staining. The number of DCX-positive cells in the hippocampus,including DG, CA1, and CA3, was significantly higher in the transgenic micethan in the nontransgenic mice.5. To determine the relative distribution of phenotypes adopted by newproliferating cells in the hippocampus, mice were injeted with BrdU at 9 monthsand sacrificed 28 days after the last BrdU inj ection. The phenotypes of newproliferating cells in the DG, CA1, and CA3 regions include neuron （BrdU+NeuN double-positive cell） and glia （BrdU + GFAP double positive cell）. Theproportion of differentiating BrdU-positive cells in transgenic and nontransgenicmice was similar, but the number of existing hippocampal BrdU-positive cellsand neurogenesis was higher in the transgenic mice.In summary, our findings are consistent with human postmortem brainstudies of AD patients that show increased hippocampal neurogenesis in DG andCA1. This study is the first to report on the changes of neurogenesis inAPP_K670N/M671N/PS1_M146L transgenic mice. As the APP/PS1 strain showsincreased neurogenesis in the progressive stage of AD, it may better reproducethe neurogenesis that is characteristic of AD patients. Although the function ofneurogenesis in the hippocampus of transgenic mice under physiological orpathological conditions is unknown, new neurons from the adult humanhippocampus have already shown some function. The formation of some types of memory relies on the continuous production of new hippocampal neuronsthroughout adulthood. Therefore, the increased neurogenesis in the ADtransgenic mice may be a compensatory response to pathological and behavioralimpairments Otherwise, new neuron may be not involved to neural network orshow some function. Although neurogenesis was increased in the hippocampus ofthe AD transgenic mouse model, the pathological change still worsened andmemory impairment could not be reversed over time. This may be due to theamount of increased neurogenesis not being sufficiently large to compensate forthe progressive pathological changes in the AD brain. Therefore, up-regulatingneurogenesis in the AD brain may be a potential therapeutic strategy for ADtreatment.更多还原