【作者】 李志方；

【导师】 李露斯；

【作者基本信息】 第三军医大学 ， 神经病学， 2008， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease,AD)是一种多发于老年人的以进行性认知功能障碍和记忆损害为主的中枢神经系统退行性疾病[1]。其病理学特征为脑内细胞外淀粉样蛋白(amyloid peptides, Aβ)斑块沉积、细胞内神经纤维缠结(Neural fibrillary tangles,NFTs)以及神经元丢失。AD的病因和发病机制十分复杂,占主导地位的Aβ学说[2]认为β-淀粉样蛋白前体(APP)裂解产生的Aβ是AD患者脑内老年斑(SP)的主要成分,它引发AD的病理改变,导致神经原纤维缠结(NFT)形成、神经细胞丢失、血管损伤和痴呆形成。目前AD的治疗仍以药物为主,包括作用于胆碱能系统的药物、抗氧化药物、抑制Aβ形成和聚集的药物、抗炎药物、5-HT受体拮抗药、钾通道阻滞剂、脑代谢激活剂及中药等[3]。由于这些药物的治疗作用主要集中在维持退化的胆碱能神经元功能及疾病的早期,对于神经元大量丢失的晚期AD患者没有治疗效果。寻找能够改善AD患者中、晚期症状和细胞丢失的治疗方法已迫在眉睫。神经干细胞(neural stem cells,NSCs)移植为AD的治疗带来了希望。神经干细胞具有自我增殖和神经分化的生物学特性,可以分化为成熟的神经细胞。通过移植外源性NSCs可望补充替代因疾病或损伤而丢失的神经元和胶质细胞[4]。这种神经替代策略已在帕金森病[5,6]、亨廷顿病[7]、卒中[8]、脊髓损伤[9]等领域取得了令人鼓舞的成绩,部分成果已成功运用于临床。文献报道,将人NSCs移植到24月龄大鼠的脑内,能有效改善老龄鼠的认知功能障碍[10]。Wang等将胚胎干细胞来源的神经球移植到AD小鼠的前额叶和顶叶皮层,移植的神经球能存活、迁移并分化为胆碱乙酰转移酶阳性神经元和少数5-羟色胺能神经元,并显著改善AD小鼠的工作记忆能力[11]。神经干细胞移植成功的关键在于植入的神经元能整合到受者神经环路中去,即与受者神经元之间形成有功能的突触联系[12]。虽然有大量的研究表明细胞替代治疗能改善动物模型的认知能力,但是对于移植细胞与受者神经环路功能性整合这一重要问题缺乏证据支持。为此,我们将胚胎干细胞(embryonic stem cells, ESCs)无血清诱导为神经前体细胞(Neural precursor cells,NPCs)后移植到Aβ1-40损伤大鼠海马齿回,观察移植细胞的迁移、分化、与受者神经系统的整合以及对AD大鼠学习记忆功能的改善情况,为AD的治疗提供实验依据。材料与方法1.本研究采用海马齿回定位注射老化态Aβ1-40的方法建立AD大鼠模型,并对该模型进行Morris水迷宫行为学测试和包括刚果红染色、Fluoro-Jade B染色、尼氏染色以及GFAP免疫组化等组织病理学鉴定。2.从E13.5胎鼠原代分离小鼠胚胎成纤维细胞,传代培养并经丝裂霉素C处理后作为饲养层细胞。小鼠胚胎干细胞体外扩增后经改良的无血清培养法诱导分化产生神经前体细胞,并对其行Nestin免疫荧光染色鉴定和分化潜能的鉴定。3.将无血清诱导的神经前体细胞定点移植到Aβ1-40损伤的大鼠海马齿回,观察移植细胞的存活、迁移、分化、整合以及细胞移植对AD大鼠学习记忆的改善情况。结果1.通过海马内注射老化态Aβ1-40多肽成功建立AD大鼠模型。该模型空间学习记忆能力明显受损。在注射区可检测到刚果红染色阳性斑块。颗粒细胞层背侧带及齿回神经元大量变性坏死、丢失,损伤区有长达4个月之久的胶质细胞反应。因此该模型能较好的模拟AD的行为学和病理学特征,损伤部位明确,制作简单,有利于进行AD的基础和细胞移植研究。2.以经丝裂霉素C预处理的小鼠胚胎成纤维细胞作为饲养层,结合1000IU/ml的LIF,能有效地维持ESCs的增殖能力和未分化状态。采用改良的无血清选择性诱导法,依次经历ESCs—EBs—NPCs等几个阶段,可获得大量高纯度的神经前体细胞。3. NPCs海马内移植能显著改善Aβ损伤大鼠的空间学习记忆能力。4. NPCs移植Aβ损伤大鼠海马齿回后向颗粒细胞层两侧发生迁移。移植细胞在体存活率较低,且随着移植时间的延长,细胞存活率逐渐降低。存活的NPCs以星型胶质细胞分化为主,少数分化为Tuj1阳性神经元。5.少数植入Aβ损伤大鼠海马齿回的NPCs能分化为谷氨酸能和γ-氨基丁酸能神经元,表达NMDA受体、AMPA受体和GABAA受体,并且在其胞膜上观察到PSD95的表达以及受者神经元来源的突触素阳性颗粒。部分分化神经元能与受者自身神经元形成突触并整合到受者神经环路中去,参与大鼠的学习记忆过程。更多还原

【Abstract】 Alzheimer’s disease (AD) is a progressive neurodegenerative disease that mainly impairs central nervous system and clinically characterized by progressive memory loss, cognitive decline in elderly population. It is the most common form of senile dementia. The main pathological changes are extracellular deposit ofβ-amyloid peptides, intracellular neurofibrillary tangles, synaptic loss, and brain atrophy. The etiology and mechanism of AD are not clear, but the amyloid cascade hypothesis is widely accepted. The hypothesis presumes that gene mutation or other factors upregulate beta-amyloid (Aβ) and disorder its metabolism, and Aβdeposits as fibril aggregates forming senile plaques (SP) and intracellular neurofibrillary tangles (NFTs), which results in the degeneration and necrosis of neurons. Current therapies, such as treatment with acetylcholinesterase inhibitors to enhance cholinergic function, give only partial and temporary alleviation of symptoms, and would not retrieve the neural loss in the cortex and hippocampus of patients at advanced stage. Neural transplantation is regarded as an inspiring strategy for the treatment of AD.Cell replacement is a promising approach for treating neurodegenerative disorders that may overcome some of the existing limitations of traditional pharmaceutical approaches. Such neuroreplacement strategies offer great therapeutic potential for the treatment of neurological diseases such as Parkinson’s disease, Huntington’s disease, spinal cord trauma, and stroke. In one study the human undifferentiated NSCs were injected into the brain of 6-month-old and 24-month-old rats respectively. Their results demonstrated that human neural stem cells improved cognitive function of aged brain. Wang and colleagues transplanted ES cells-derived neurospheres into mouse model frontal cortex of Meynert nucleus lesion. They found that transplanted neurospheres survived, migrated and differentiated into many choline acetyltransferase-positive neurons and a few serotonin-positive neurons. The working memory error decreased significantly in the mice grafted with neurospheres. Despite the broad experimental application of neuronal transplantation, few studies have addressed the functional integration of single neurons in the host CNS. Therefore, our purpose focus on the differentiation and integration of engrafted NPCs after transplanted into the hippocampus of Aβ1-40-injured rats.Materials and methods:1. Rats model was established by the intrahippocampal injection of aggregatedβ-amyloid (1-40), the learning and memory of AD model rats was measured by the Morris water maze test. The histopathological changes of AD model was observed by Congo Red staining, Nissl staining, Fluoro-Jade B(FJB) staining and GFAP immunohistochemistry.2. Mouse embryonic fibroblast (MEF) were separated from E13.5 pregnant mouse and passaged 3-5 generation, then used as feeder layers treated by mitomycin C for 150 minutes,. An EGFP-expressing derivative of the ES cell line MESPU35 were expanded on feeder layers combinated with leukemia inhibitory factor (LIF). NPCs were generated from ESCs by the modified serum-free methods, and detected by Nestin immunohistochemistry.3. NPCs generated from mouse ESCs in vitro were transplanted into the hippocampus of Aβ1-40-injured rats. And the survive, migration, differentiation and integration of engrafted NPCs were observed, and the improvement of memory dysfunction of Aβ1-40-injured rats was also observed.Results1. The AD model was established by intrahippocampal injection of aggregated beta-amyloid(1-40). Learning and memory of the AD rats were significantly declineded by Morris Water Maze test two weeks post-surgery. The Congo Red-positive plaques were detected around the injection sites of Aβ1-40. FJB and Nissl staining showed the obvious neural degeneration and loss around the injection site. Furthermore, the GFAP-positive astrocyte were over-activated compared with NS-injected control group for four month longer. This model can mimic the cognitive functions impairment and pathology characters of AD well and facilitate the following research on NPCs transplantation.2. Purified mouse embryonic fibroblasts (MEF) were isolated from E13.5 pregnant mouse to prepare feeder layer for ES culture. ES cells can be expanded in vitro without differentiation under the condition of feed layer combinated with leukemia inhibitory factor (LIF). After removal of feeder cells, hLIF and planted on the bacterial culture dishes, embryonic bodies were formed, and then differentiated into NPCs under the serum-free medium supplemented by N2 plus fibronectin, about 93% of which were Nestin-positive cells.3. After intrahippocampal transplantation of NPCs, the cognitive functions of AD rats were assessed by Morris water maze test. It shows learning and memory impairment of AD rats was improved significantly at 4w、8w、12w、16w post-transplantation.4. Engrafted NPCs migrated steadily farther under the guidance of local circumstance as time goes on. The survival rate of engrafted NPCs was 7.32±0.69% and decreased significantly gradually from 4w to 16w. At the same time, the living NPCs mostly differentiated into GFAP-positive astrocytes and some differentiated into Tuj1-positive neurons.5. A few engrafted NPCs expressed the neuronal glutamate transporter protein (EAAT3) and the rate-limiting enzyme for GABA synthesis (GAD67), suggesting efficient differentiation into glutamatergic neurons and GABAergic neurons. Some engrafted NPCs express ionotropic glutamate and GABA receptors using antibodies to the AMPA receptor (GluR1), the GABAA receptor (βchain), and the NMDA receptor (NR1). Confocal immunofluorescence analysis revealed PSD95-positive puncta were typically found in close proximity to the engrafted NPCs surface and numerous synaptophysin-positive, EGFP-negative patches were found in close apposition to the somatic and dendritic membranes of transplanted cells, suggesting that host-derived presynaptic terminals contact incorporated NPCs-derived neurons. Ultrastructural analysis demonstrated the synapse formation between the donor cell-derived neurons and the host neurons. Morris water maze test bombined immunohistology of Fos-expression indicate some engrafted NPCs-derived neurons incorporated into the host brain circuitry and participated the learning and memory process.更多还原