针对β-分泌酶1适配子的筛选及其联合AChE的适配子治疗AD的实验研究

Suppression of the formation of amyloid-beta (Aβ) and its toxicity is the fundamental strategy in the treatment of Alzheimer's disease (AD). β-site amyloid precursor protein cleaving enzyme 1 (BACE1) plays a crucial role in the process of Aβ generation. Consequently, one way to prevent Aβ generation from the source maight be to inhibit the activity of BACE1. The systematic evolution of ligands by exponential enrichment (SELEX) technology, which can select inhibitors that bind to their target proteins with high specificity and good affinity, is considered a new method of drug screening. In this study, we aim to adopt SELEX technology to select aptamers able to inhibit the extracelluar region of BACE1 activity specifically. Currently acetylcholine esterase (AChE) inhibitors are the most common clinical drugs for the treatment of AD. Based on the consensus that "multi-target combination therapy" can improve therapeutic effect because of the multi-factor pathogenic mechanism of AD and the fact that anti-AChE aptamers have been selected in our previous report, we attempt to evaluate the effect of anti-BACE1 aptamer alone or combined with anti-AChE aptamer on cells stably transfected with APPsw or APPwt genes and on Aβ production in these cellular models of AD. Next we try to incorparate these aptamers into nanoparticles to observe its influence on behavior changes and pathological changes of Aβ and senile plaque generation in AD animal models. Simultaneously we will investigate the role of anti-BACE1 aptamer on early diagnosis of AD. In our study SELEX technology is applied to explore new approach and ideas for AD diagnosis and treatment, which provides a new technology platform for correlational research of AD.

对抗Aβ形成及其毒性是治疗AD的一种根本策略。BACE1是Aβ生成过程中的关键酶，抑制BACE1活性可从源头上阻断Aβ的产生。SELEX技术可筛选出与靶蛋白的高特异和高亲和力结合的抑制剂，是一种新型的药物筛选方法。本课题采用SELEX技术筛选抑制BACE1胞外区蛋白活性的适配子（aptamer）。目前最常用药物就是AChE抑制剂，"多靶标联合用药"可提高AD治疗效果。我们已筛选出具有抑制作用的AChE适配子。所以我们尝试将BACE1适配子单独或与联合AChE适配子分别作用于稳定表达APPsw、APPwt的AD细胞模型，观察其对细胞的作用和Aβ产量的影响；将适配子与纳米颗粒结合，观察其对AD动物模型的行为学改变及脑内Aβ和老年斑生成等变化。同时还探讨BACE1适配子在AD早期诊断中的作用。本课题运用SELEX技术来进行探索诊断和治疗AD的新方法和新思路，为AD诊疗相关基础研究提供新的技术平台

BACE1是Aβ生成过程中的关键酶，抑制BACE1活性可从源头上阻断Aβ的产生。SELEX技术可筛选出与靶蛋白的高特异和高亲和力结合的抑制剂，是一种新型的药物筛选方法。本课题采用SELEX技术筛选抑制BACE1蛋白活性的适配子（aptamer）。目前最常用药物就是AChE抑制剂，“多靶标联合用药”可提高AD治疗效果。主要取得以下创新性成果：（1）建立了一种实用的BACE1表达、纯化和鉴定方法，为靶向BACE1抑制剂的研制及后续实验提供研究基础；（2）采用微孔板SELEX筛选得到以高亲和力特异结合BACE1的适配子，并进行克隆测序和二级结构预测。适配子与BACE1的Kd值在nM水平。用pull down实验验证了适配子A1可结合BACE1蛋白。（3）利用荧光共振能量转移实验检测BACE1特异的适配子A1在体外和AD细胞模型中可抑制BACE1的活性。A1的IC50为139.81nM；（4）通过水迷宫等动物行为实验证明BACE1特异的适配子A1可改善AD动物模型Tg6799的学习记忆能力。适配子A1处理组的小鼠脑内板块沉积数量明显少于对照组，小鼠海马内Aβ40和Aβ42含量的明显减少，BACE1和sAPPβ的表达量显著降低。（5）我们对AChE适配子Ob2进行了抑制AChE活性检测，发现Ob2可以很好地抑制小脑匀浆和rhAChE的酶活性。（6）采用脑内埋管给药的方法进行Ob2、Ob2+A1、A1、scr分组，给药后进行行为学检测和WB实验。结果显示Ob2单独给药和对照组相比有很好的提高小鼠记忆能力的作用；适配Ob2和适配子A1在实验中具有协同作用，为我们的“多靶标联合用药”思路提供有力证据。（7）我们还尝试了用PLGA纳米粒子包裹适配子进行脑外给药的初步研究。对负载适配子的PEG-PLGA进行了表征和鉴定。制备的纳米粒子具有穿透血脑屏障的作用，我们进一步进行了AD的治疗(这部分数据正在整理中)。（8）BACE1适配子用于AD诊断的研究结果提示BACE1适配子有可能作为早期诊断试剂用于AD的诊断。以上研究结果对于建立成功SELEX的方法、适配子特性鉴定及其体内外作用、适配子介导的纳米粒子的制备等方面提供研究经验。本课题运用SELEX技术来进行探索诊断和治疗AD的新方法和新思路，为AD诊疗相关基础研究提供新的技术平台。