阿尔茨海默病早期诊断生物标志和基因重组疫苗的研究

【作者】 张建民；

【导师】 何维； 崔莲仙；

【作者基本信息】 中国协和医科大学 ， 免疫学， 2003， 博士

【摘要】 阿尔茨海默病（Alzheimer’s disease，AD）是一种常见的中枢神经系统进行性变性疾病，其主要临床表现为进行性记忆减退和认知障碍。目前临床上缺乏辅助AD早期诊断的特异性生物学标志，也没有有效的防治手段。我们主要在这两方面进行了研究。一、AD早期诊断生物标志的研究目前人们发现AD患者存在多种生物学指标发生异常，但是大多数缺乏特异性或敏感性。近几年研究发现，AD患者存在不同程度的免疫系统功能损伤，而且这种免疫功能紊乱参与了AD的病理发生及发展过程。我们从免疫学角度入手分析了AD病人的若干项免疫学指标，以期寻找到相对特异和敏感的生物学指标，辅助临床对AD的早期诊断，并初步探讨了AD病人免疫功能损伤的可能机制。首先，我们检测了32例AD患者和32例健康对照外周血中细胞因子IL-1β，IL-6，TNF-α和M-CSF的水平。结果显示，AD患者外周血中M-CSF的水平明显高于健康对照者。为了进一步研究AD患者外周血M-CSF高表达的特异性以及其与AD患者痴呆程度间的相互关系，我们分析了180例AD患者、53例血管性痴呆（Vascular dementia，VD）患者和92例健康对照外周血中M-CSF的水平。结果显示，AD患者外周血中M-CSF的水平明显高于VD病人组和健康对照组，而且外周血中M-CSF的水平与AD患者的痴呆程度之间具有明显的正相关性；而VD患者外周血中M-CSF的水平与健康对照组相比则无明显的统计学差异。上述结果表明M-CSF参与了AD的病理发生发展过程并发挥重要作用。其次，我们从AD患者存在免疫功能损伤的角度入手，探讨AD患者免疫功能紊乱的机制。为此，我们检测了187例AD患者、53例VD患者和80例健康对照者外周血单个核细胞（PBMC）经植物血凝素（PHA）活化培养后的端粒酶活性水平。结果显示，AD患者PHA活化的PBMC的端粒酶活性明显高于健康对照组，并且端粒酶活性的水平与AD患者痴呆程度之间具有明显的正相关性；相反，VD患者PBMC的端粒酶活性与健康对照组相比较，没有明显的统计学差异。从AD患者PBMC的增殖实验发现，其增殖能力明显低于健康对照组。由于淋巴细胞端粒酶活性水平的高低反映了其端粒稳定性的情况，所以，这些结果提示AD患者PBMC的端粒存在不稳定性，功能发生障碍，从而激发了细胞端粒酶的活性水平升高。这也许就是造成AD免疫功能损伤的重要机制之一。综上所述，M-CSF在AD的病理发生及发展过程中发挥了重要的作用。PBMC尤其是T细胞端粒的功能障碍可能是造成AD病人免疫功能损伤的重要原因之一。AD病人外周血中M-CSF的水平和PBMC的端粒酶活性可以作为辅助AD早期诊断和监测AD患者免疫功能紊乱的生物学指标。二、AD基因重组疫苗的研究AD的主要神经病理学特征包括海马和大脑新皮质区神经元的弥散性丢失、细胞内神经纤维缠结和细胞外淀粉样蛋白的沉积。淀粉样蛋白沉积斑的主要成分是Aβ（amyloidβpeptides），Aβ是一组由β淀粉样前体蛋白（β-amyloid precursor protein，APP）降解产生的含39～43个氨基酸残基的混合物。目前研究证实，脑组织内Aβ的积聚是AD病理发生的最初原因。AD的其它病理过程包括含tau蛋白的神经纤维缠结的形成等均被认为是由于Aβ的生成与清除之间发生失衡引起的。因此，目前世界上有相当多的科学家均致力于研发能够改善Aβ生成与清除平衡的治疗手段。在本研究中，我们构建了一种霍乱毒素B亚单位-Aβ42基因重组腺相关病毒疫苗（AAV-CB-Aβ42），并采用了多种免疫途径观察了该疫苗对PDAPP^V7171转基因小鼠的药效学作用。研究结果显示，该疫苗在体内能够表达霍乱毒素B亚单位与Aβ42肽结合的融合蛋白，该融合蛋白能够刺激机体产生抗Aβ抗体。一次性免疫PDAPP^V7171转基因小鼠能够产生长期有效的Aβ特异性IgG，而且能够改善小鼠的记忆和认知行为能力，降低脑组织内Aβ沉积和反应性星形胶质细胞的活化程度。我们的实验结果拓宽了治疗或／和预防AD的方法，为AD的防治提供了新思路。同时，我们的实验结果显示AAV-CB-Aβ42疫苗有望开发成一种新型的、有效的AD防治疫苗。更多还原

【Abstract】 Alzheimer’s disease （AD） is a progressive degenerative disease, which is characterized clinically by a progressive loss of memory function and cognition impairment associated with the presence of degenerative well known pathological lesions. There are no specific biomarkers for assisting the early diagnosis of AD and no efficient approaches for preventing or treating AD. The present study included two principal issues as follows. 1. The study of biomarkers for assisting early diagnosis of ADMany biomrkers have been found to be associated with the development of AD. However, most of them seem to be lack of specificity or sensitivity. The pathogenesis of AD is unclear so far. The involvement of the immune system in the pathogenesis of AD has been studied for years. Recent reports demonstrated that there was impairment of immune function in patients with AD, and the perturbation of immune system was involved in the pathogenesis of AD. In the present study, we analyzed several immunological biomarkers of AD patients in order to search certain specific or sensitive biomarkers for early diagnosis of AD, and primarily investigated the possible mechanism of the impairment of immune function in AD patients.First, we analyzed the levels of IL-1, IL-6, TNF-αand M-CSF in sera from 32 cases of AD patients and 32 cases of age- and sex-matched healthy controls. The results demonstrated that levels of M-CSF in sera in AD patients significantly increased when compared with healthy controls （P<0.05）. To further analyze the specificity of overexpression of M-CSF in patients with AD and the correlation between the levels of M-CSF and the degree of dementia of AD patients, we analyzed the levels of M-CSF in sera from 180 patients with AD, 53 patients with Vascular dementia （VD）, and 92 age- and sex-matched healthy controls, respectively. The results showed that the levels of M-CSF in sera from AD patients were significantly increased when compared with VD patients and healthy controls, respectively, and there was significantly statistical correlation between the levels of M-CSF in sera from patients with AD and the degree of dementia of patients with AD. In contrast, no significantly statistical difference was found between the levels of serum M-CSF of VD patients and that of healthy controls. These data indicate that M-CSF might be involved in the pathologic process.Second, we explored the mechanism of the perturbation of immune function in patients with AD. We analyzed telomerase activities of PHA-activated peripheral blood mononuclear cells （PBMC） from 187 cases of patients with AD, 53 cases of patients with VD, and 80 cases of age-matched healthy controls, respectively. The results showed that telomerase activities of PHA-activated PBMC in AD patients were significantly much higher than those in healthy controls or VD patients, respectively, whereas no significant difference was found between the telomerase activities of PHA-activated PBMC in AD patients and those of healthy controls. We also found that there was significantly statistical correlation between the telomerase activities of PHA-activated PBMC and the degree of dementia in AD patients. Since the changes of telomerase activity reflect the instability of telomere, these data indicate that telomere instability of PBMC occurs in patients with AD, and the dysfunction of telomere contributes to the increase of telomerase activities of PBMC.In summary, our findings indicated that M-CSF might be involved in the pathogenesis of AD, and play an important role in the development of neuropathological changes of patients with AD, while the dysfunction of telomere of PBMC, especially T lymphocytes, could contribute to the impairment of immune function in AD patients. Telomerase activities of PBMC and M-CSF might be useful biomarkers for early diagnosis of AD and monitoring the perturbation of immune function of AD.2. The study of gene recombinant vaccine against ADAlzheimer’s disease （AD） is a neurodegenerative disorder characterized by a progressive decline of cognitive abilities and by neuropathological features’ including diffuse loss of neurons in the hippocampus and neocortex, accumulation of intracellular protein deposits （neurofibrillary tangles）, and accumulation of extracellular protein deposits （amyloid or senile plaques） characteristically seen in the associative cortices and limbic system. A main constituent of these amyloid plaques is the amyloid-β-peptide （Aβ）, a 39-43-amino-acid protein derived from the processing of a large transmembrane protein, theβ-amyloid precursor protein （APP）. It has been postulated that accumulation of Aβin the brain, resulting from abnormal processing of APP to Aβor reduced clearance of Aβfrom brain, is the primary cause of AD pathogenesis. The rest of the disease process, including formation of neurofibrillary tangles containing tau protein, is thought to result from an imbalance between Aβproduction and Aβclearance. As a result, there is a widespread interest in developing therapeutic approaches to improve the balance between Aβproduction and Aβclearance.In the present study, we developed a cholera toxin B subunit （CB） and Aβ42 gene-recombinant adeno-associated virus （AAV-CB-Aβ42） vaccine and detected the efficacy of it on PDAPP^V7171 transgenic mice with different routes of administration. The results showed that the AAV vaccine expressed a fusion protein of CB and Aβ42 in vivo. A single administration of the AAV-CB-Aβ42 vaccine induced a prolonged, strong production of Aβspecific serum IgG in PDAPP^V7171 transgenic mice, and resulted in improved ability of memory and cognition, decreased Aβdeposition in the brain and a resultant decrease in plaque-associated astrocytosis. Our results extended the immunological approaches for the treatment and prevention of AD to an oral, intranasal or intramuscular route that might be better tolerated in human patients than repetitive immunizations in the presence of adjuvant. Our data raised the possibility that AAV-CB-Aβ42 vaccine immunization may provide the basis of a novel and promising Alzheimer’s disease vaccination strategy.更多还原