【作者】 武强；

【导师】 李露斯；

【作者基本信息】 第三军医大学 ， 神经病学， 2007， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease, AD)是发生于老年和老年前期以进行性认知障碍和记忆力损害为主的中枢神经系统退行性疾病。随着分子生物学和分子病理学的飞跃发展,AD的病因及发病机制的研究取得了很大进展,淀粉样蛋白级联反应被认为在AD的发病机制中占主导作用,已成为AD发病机制研究中的热点和治疗AD的突破点。淀粉样蛋白级联反应学说认为基因突变或其它因素使Aβ淀粉样蛋白(amyloidβprotein, Aβ)的表达增加或代谢紊乱,导致Aβ的产生和清除不平衡,日积月累造成Aβ的沉积,形成老年斑(senile plaque, SP)和神经原纤维缠结(neurofibillary tangles, NFTs),最终导致神经细胞变性坏死。越来越多的资料表明Aβ淀粉样肽产生过多,其聚集物在脑组织中的形成与沉积是AD发病的一个中心环节和共同通道。因此,若能够减少Aβ肽的形成、抑制Aβ肽的聚集、抑制Aβ多肽的神经毒性、加速Aβ肽的降解与清除,则可能成为预防和治疗AD的理想途径。目前AD常规的治疗,既不能消除Aβ的沉积,也没有改善神经元的丢失。难以达到理想的治疗效果。随着基因治疗和干细胞工程技术的迅猛发展,为解决这一难题提供了新的思路。α2巨球蛋白(Alpha-2 macroglobulin, A2M)是一泛蛋白酶抑制剂,也是脑组织免疫炎症反应的一种急性期反应蛋白,主要由肝脏合成,在脑内可由星形胶质细胞产生,它广泛分布在细胞外液中,在血浆中维持较高的浓度。近来研究发现,A2M是Aβ肽的一种高亲和力的结合蛋白,其C末端的27个氨基酸可与Aβ肽特异性结合。通过A2M与Aβ的相互作用,A2M可中和Aβ的毒性、降解并清除Aβ。另外,A2M还可与一些小分子如细胞因子、生长因子、免疫炎症因子等结合并清除这些分子,从而影响细胞的表型,影响细胞因子、生长因子、免疫炎症因子等的平衡以及局部微环境的稳定,调节细胞的生长和生理过程。通过不同的限制性内切酶消化及聚合酶链反应(Polymerase chain reaction, PCR)方法可将A2M分成6个片段,即FP1(aa99-392),FP2(aa341-590),FP3(aa591-744),FP4(aa775-1059),FP5(aa1030-1279),FP6(aa1242-1451)。各片段与生长因子、Aβ的体外试验证明,以转化生长因子( transforming growth factor-beta, TGF-β)为代表的生长因子选择性与FP3段结合,Aβ则与FP6段选择性结合,且其结合具有高度的特异性。这也提示A2M(FP6)可能成为一个治疗AD新的方向。神经干细胞(Neural Stem Cells,NSCs)作为神经系统的全能细胞,有很强的分裂、增殖能力,且在不同因子作用下,定向分化成神经元和神经胶质细胞。体外培养的神经干细胞作为神经损伤、修复的治疗性供体已经展现了诸多的优点。而且它也是非常理想的基因治疗载体。鉴于A2M具有中和Aβ的毒性、降解并清除Aβ及其FP6段能特异性结合Aβ的重要特性,利用NSCs可作为基因治疗的载体及其对缺失神经元的替代作用,本研究小组在成功构建了pEGFP/A2M(FP6)真核表达载体的基础上,应用脂质体转染获得转染pEGFP/A2M(FP6)的NSCs,探索转染pEGFP/A2M(FP6)的NSCs治疗AD的新方法。初步实验发现,pEGFP/A2M(FP6)的转染能明显提高体外培养的NSCs体系对Aβ毒性的耐受性,并减少Aβ处理后细胞的死亡。在此基础上,本实验采用PCR、NSCs分离培养、基因转染等技术,并结合水迷宫测试、转基因小鼠鉴定、定向海马移植、免疫组化染色以及荧光显微镜观察和图像分析等手段,首先对所获pEGFP/A2M(FP6)重组质粒进行鉴定,将构建好的pEGFP/A2M(FP6)真核表达载体转染至NSCs中,观察携带pEGFP/A2M(FP6)NSCs的生物学特性,并将其定向移植到APP/PS1双转基因AD小鼠海马内,观察移植细胞的存活、分化、迁移,脑内Aβ沉积的变化,以及对APP/PS1双转基因AD小鼠学习记忆功能的影响。结果如下:一、pEGFP/A2M(FP6)重组质粒转染神经干细胞的实验研究1.采用机械分离法,成功的分离出胚胎小鼠皮层NSCs,利用DMEM/F12(1:1)培养基,加入生长因子B27和碱性成纤维细胞生长因子( basic fibroblast growth factor, bFGF),成功地进行了NSCs的培养和传代。采用Nestin、GFAP、MAP-2进行鉴定,并进行了MAP-2和GFAP的双标记,培养的细胞有阳性表达,充分说明我们分离、培养的细胞为NSCs,而且具有向神经元和神经胶质细胞分化的潜能。NSCs分离、培养、鉴定的完成为pEGFP/A2M(FP6)转染提供了载体。2.利用Nucleofector转染技术成功将pEGFP/A2M(FP6)重组质粒转染入NSCs,转染率约为50%,经G418筛选传3代,荧光显微镜下仍可见EGFP的表达,经10%胎牛血清诱导后,免疫荧光组织化学染色显示部分细胞MAP-2、GFAP表达阳性。表明转染pEGFP/A2M(FP6)对神经干细胞的生长、分化无明显影响。转染pEGFP/A2M(FP6)的NSCs用RT-PCR可检测到A2M FP6mRNA的高表达,免疫荧光组织化学法观察到A2M蛋白的表达。这也为我们细胞移植治疗AD提供了理论及实验基础。二、APP/ PS1双转基因AD传代小鼠的基因型鉴定及其行为学和组织学分析1.对转基因小鼠杂合体种鼠(来源于Jackson实验室)进行繁殖,抽取小鼠的尾血提取基因组DNA,并进行基因型分析,筛选得到了APP/PS1双转基因AD小鼠。2.抗Aβ免疫荧光组织化学染色显示APP/PS1双转基因AD小鼠的皮层及海马区有大量的Aβ染色阳性斑块。透射电子显微镜下海马区Aβ周围神经元溃变、肿胀,细胞内含有大量的脂褐素,神经突触稀松形态不一,微管小泡及线粒体堆积,胶质细胞胞浆肿胀。水迷宫实验发现该小鼠逃避潜伏期较对照鼠显著延长。表明APP/PS1双转基因AD小鼠具有AD主要的病理及行为学特征,为本研究提供了理想的AD动物模型。三、携带目的基因的神经干细胞移植治疗AD小鼠的实验观察1. AD小鼠水迷宫测试,pEGFP/A2M(FP6)-NSCs组和pEGFP -NSCs组小鼠逃避潜伏期较SO组及ACSF组显著缩短;pEGFP/A2M(FP6)-NSCs组和pEGFP -NSCs组AD小鼠逃避潜伏期相比,差异有统计学意义(p<0.05)。表明两组均能有效改善AD小鼠的学习记忆障碍,以pEGFP/A2M(FP6)-NSCs组改善效果更佳。2.抗Aβ免疫荧光组织化学染色显示,pEGFP/A2M(FP6)-NSCs组和pEGFP -NSCs组AD小鼠海马及皮层内,部分Aβ染色阳性斑块周围可见表达EGFP的移植细胞,表明移植的神经干细胞可迁移并包绕Aβ斑块。观察pEGFP/A2M(FP6)-NSCs组AD小鼠额叶、海马区域,Aβ染色阳性斑块数量和平均面积均较pEGFP -NSCs组、SO组及ACSF组显著减少。表明pEGFP/A2M(FP6) NSCs移植治疗可以减少AD小鼠皮层及海马区Aβ斑块的沉积。3.移植的神经干细胞,部分细胞Nestin染色呈阳性,表明仍具有干细胞的活性,MAP-2染色显示少数移植细胞呈阳性, GFAP染色部分移植细胞呈阳性。说明部分移植的神经干细胞分化为神经元及星形胶质细胞。本实验首先对所获pEGFP/A2M(FP6)重组质粒进行鉴定,将之成功地转染入神经干细胞,使其高表达具有生物学活性的A2M(FP6)。并将转染pEGFP/A2M(FP6)神经干细胞移植到APP/PS1双转基因AD小鼠海马,观察到AD小鼠脑内Aβ斑块沉积减少,部分移植的神经干细胞分化为神经元及星形胶质细胞,小鼠的学习记忆功能显著改善。本研究从神经干细胞应用及基因治疗的角度对AD的治疗进行了有益的探索。更多还原

【Abstract】 Alzheimer’s disease (AD) is a progressive neurodegenerative disease that mainly impaires central nervous system and clinically characterized by progressive memory loss, cognitive decline in elderly population. Great advancement has been gained in etiology. The amyloid cascade hypothesis is widely accepted in AD pathogenesis. 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. Therefore, amyloid cascade hypothesis has become a hot spot on the investigation of pathogenesis and a breakthrough of the therapy on AD. Many studies show that deposits of Aβin brain is a central and common passage in pathogenesis of AD. AD can be relieved and even cured by decreasing formation, inhibiting aggregate, restraining neurotoxicity, accelerating degradation and clearness of Aβin brain.Alpha-2 macroglobulin (A2M) is a proteinase inhibitor with broad-range specificity and an acute phase reactive protein relating to immune and inflammatory reaction of brain tissues. A2M is found mainly in the liver and in astrocytes of the brain. It is widely distributed over extracellular fluid, and kept in the plasma at higher concentration. Present studies show that A2M is a high affinity protein of Aβ, of which 27 amino acids of C-terminal can specifically bind, neutralize toxicity, degrade and clean Aβ. Furthermore, A2M can also bind and clean some micromolecules, such as cytokines, growth factors, immunine and inflammatory factors. Therefore, A2M effects corpuscular phenotype and balances of cytokines, growth factors, immune and inflammatory factors, stabilizes the region microenviroment and accommodates the procedure of cells growth and physiology. A2M can be fractionated into six fusion proteins, including FP1 (aa99-392), FP2 (aa341-590), FP3 (aa591-744), FP4 (aa775-1059), FP5 (aa1030-1279) and FP6 (aa1242-1451). In-vitro studies have confirmed that growth factors (such as TGF-β, PDGF-BB, NGF-β) bind selectively FP3 and Aβbind preferentially FP6.Considering the important role that A2M can counteract toxicity of Aβ, degenerate and clean up Aβ, and FP6 can specificially bind to A2M, and neural stem cells (NSCs) can repair the absenced neuron and gene therapy as vector, our research team has successfully constructed Eukaryotic expression plasmid vector pEGFP/A2M(FP6)and transfected it into NSCs with lipotransfection in order to explore a new therapeutical means of AD. Initial experiments found that pEGFP/A2M(FP6)-transfected neural stem cells can elevate toleration of Aβtoxicity, and reduce cells death attributable to Aβ. Based on above experiments, the objective of this present study is to identify the plasmid pEGFP/A2M(FP6)and transfect it into NSCs, and then the gene-modified NSCs were transplanted into hippocampus of APP/PS1 double transgenic mice thus investigating the survivial, migration and differentiation of NSCs and the aleration of Aβin the brain of transgene mice. The main results are as follows:Ⅰ. Transfection of NSCs with pEGFP/A2M (FP6) in vitro.1. Culture and identification of NSCs.NSCs were isolated and cultivated. Immunohistochemistry showed the NSCs of passage 3 expressed Nestin, GFAP, and MAP-2, suggesting that the cells have NSCs characteristics. Further, The co-localization of GFAP, and MAP-2 confirmed NSCs. It differentiated to neuron and glia.2. The pEGFP/A2M (FP6) was successfully transfected into neural stem cells.With the aid of Nucleofector, the pEGFP/A2M (FP6) was successfully induced into NSCs with high transfection rate of 50%. Green fluorescence was observed in the transfected cells, indicating the existence of reporter gene. After screening with G418, the assay of cell proliferation and immunohischemistry showed that the proliferation of cells is unchanged and the expression of MAP-2, GFAP positive. The expression of A2M FP6 mRNA was detected with RT-PCR and electrophoresis showed that the expression of A2M FP6 in transfected NSCs was higher than that of pEGFP transfected cells. Immunofluorenscent analysis observed the expression of A2M FP6 protein. These findings suggest that pEGFP/A2M (FP6) was successfully transfected into NSCs.Ⅱ. Identification and ethology, histologic analysis of APP/PS1 double transgenic Alzheimer’s disease mice model.1. APP/PS1 double transgenic AD mice were purchased from Jackson laboratory. The animals were breeded and the offsprings were genotyped according to the guildeline of Jackson laboratory.2. Immunofluorescent assay showed that many Aβdeposits were discovered in cortex and hippocampus of APP/PS1 mice. Transmission electron microscope observed that neurons were found to be degenerating and swelling surrounding the Aβ, synapse loosing, microtube and mitochondrium accumulating, glias swelling. Water maze test showed the latencies of double transgenic APP/PS1 mice were longer than that of control group. This suggests APP/PS1 mice could simulate the specific pathogenesis of Alzheimer’s disease, thus can be regard as a efficiently experimental animal model.Ⅲ. The effect of pEGFP/A2M (FP6) transfected NSCs in transgenic mice of Alzheimer’s disease1. The pEGFP/A2M (FP6) transfected NSCs and infused into the hippocampal area of APP/PS1 mice, and water maze test was carried out at 53th-59th day. The latencies of pEGFP/A2M (FP6)-NSCs group and pEGFP-NSCs group were shorter than that of sham operated (SO) group and artificial cerebrospinal fluid (ASCF) group. pEGFP/A2M(FP6)- NSCs group showed the best improvement, its mean latencies were significantly shorter than pEGFP -NSCs group (p<0.05) and learning and memory were improved.2. Anti-Aβdetection showed Aβdeposits in hippocampal and cortex of pEGFP/A2M (FP6)-NSCs group and pEGFP- NSCs group were surrounded by transplanted NSCs. The amount of Aβdeposits and average size of Aβdeposits in hippocampus and cortex of pEGFP/A2M(FP6)-NSCs group were reduced markedly, compared with that of sham operated group, ASCF group and pEGFP -NSCs group(p<0.05) .This suggests pEGFP/A2M(FP6)-NSCs can reduced Aβdeposits in hippocampus and cortex of AD mice.3. The expression of Nestin protein were still detected positively 2 months after transplantation, and this manifested they still had stem cells potential ability. Immunofluorescent detection indicated that majority of transplanted cells were positively expressed GFAP while only a few espressed MAP-2. This findings suggest the transplanted NSCs were differentiated into neurons and glias.In conclusion, we first identified pEGFP/A2M (FP6) and transfected it into NSCs to obtain the seed cells with efficient expression of A2M (FP6), and then transplanted the transfected NSCs into the hippocampal district of APP/PS1 double transgenic mice. Partial transplanted NSCs were differentiated into neurons and glias, Aβdeposits were reduced, and the function of learning and memory were promoted. This study lay the foundation on developing new approaches to treat AD.更多还原