抗Aβ31-35单克隆抗体在阿尔茨海默病APP/PS1转基因鼠中的实验研究

The cognitive impairments in Alzheimer disease (AD) are closely related to the deposition and neurotoxicity of amyloid β protein (Aβ) in the brain. Although the immunotherapy aimed at clearing Aβ obtained remarkable achievements in recent years, the clinical trials of AD immunotherapy have been suspended because of serious side effects of anti-Aβ antibody, in which the physiological amyloid precursor protein (APP) was mistakenly attacked by anti-Aβ antibody. Therefore, it is very critical to prepare a specific antibody against Aβ but without any hurt to APP. Based on our previous finding that the sequence 31-35 of Aβ is the active center of Aβ molecule, the present study will prepare a specific monoclonal antibody against sequence 31-35 of Aβ by using phage display peptide library technology. On this basis, we will investigate the neuroprotective effects of anti-Aβ31-35 monoclonal antibody against the cerebral pathological impairments and cognitive deficits seen in APP/PS1 transgenic mouse. Further, the cellular and molecular mechanisms of anti-Aβ31-35 monoclonal antibody will be examined by recording in vivo hippocampal field potential, brain slice ion channel currents, intracellular calcium level and cerebral inflammatory factors in the brain. Overal, the study will try to provide a novel, safer and more effective immunotherapeutic strategy for the prevention and treatment of AD.

阿尔茨海默病（AD）认知功能下降与患者脑内淀粉样β蛋白（Aβ）的聚集和毒性密切相关。近年来以清除Aβ为目的的免疫疗法令人瞩目，但临床试验却因严重副作用而暂停，这与细胞膜上生理性淀粉样前体蛋白（APP）受到错误攻击有关。因此，设计和使用既能有效清除Aβ又不伤害APP的特异性抗体有望突破AD治疗瓶颈。本课题在以往确认Aβ活性中心的基础上，将巧妙利用31-35序列在APP结构域上的分布特征，采用基因工程噬菌体展示肽库技术制备选择性结合Aβ而规避APP的抗Aβ31-35单克隆抗体；将利用APP/PS1转基因鼠，研究抗Aβ31-35单克隆抗体预防和逆转AD动物病理性脑损伤及认知功能下降的神经保护效应；并通过检测AD转基因鼠在体海马场电位、脑片离子通道电流、培养细胞钙水平和脑内炎性反应因子等，阐明抗Aβ31-35单克隆抗体神经保护效应的细胞和分子机制。从而为AD防治提供一种安全、有效和新颖的免疫策略。

阿尔茨海默病（AD）认知功能下降与患者脑内淀粉样β蛋白（Aβ）的聚集和毒性密切相关。近年来以清除Aβ为目的免疫疗法令人瞩目，但临床试验却因严重副作用而暂停，这与细胞膜上生理性淀粉样前体蛋白（APP）受到错误攻击有关。因此，设计和使用既能有效清除Aβ又不伤害APP的特异性抗体，有望突破AD的治疗瓶颈。本研究在以往确认Aβ活性中心的基础上，巧妙利用31-35序列在APP结构域上的分布特征，采用基因工程噬菌体展示肽库技术制备了选择性结合Aβ而规避APP的抗Aβ31-35单链抗体scFv17；利用APP/PS1转基因小鼠，通过了行为学、在体海马场电位记录以及免疫组织化学等方法，研究了抗Aβ31-35单链抗体逆转AD动物认知功能下降、逆转AD动物病理性脑损伤以及脑内炎性反应因子等神经保护效应。实验结果表明：抗Aβ31-35单链抗体在体外能够同等程度地与Aβ31-35和Aβ1-42结合；scFv17能够逆转在APP/PS1转基因小鼠的空间学习和工作记忆；在脑内已经形成Aβ斑块的APP/PS1小鼠脑内注射scFv17一周后，能够明显减少AD模型鼠脑内的Aβ沉积。鼻饲scFv17抗体12周后能够明显减轻和减少AD模型鼠脑内星形胶质细胞和小胶质细胞的增生；鼻饲scFv17抗体可以促使AD模型鼠脑内IL-1β、IL-10、TGF-β的增加，而使表达增加的TNF-α减少。这些结果证实了抗Aβ31-35单链抗体在治疗AD动物中的有效性。对淀粉样蛋白假说提供了有力的支持。提示该抗体有可能成为一种新的安全、有效的AD免疫治疗策略。