基于microRNA调控叶酸对转基因AD小鼠神经干细胞作用机制的研究

There is no specific clinical treatment for Alzheimer's disease (AD) so far. Seeking the methods to stimulate endogenous neural stem cells (NSCs) to treat AD is one of the research hotspots at present. We previously found that folic acid can promote NSCs proliferation and differentiation. However, the impacts of folic acid on neurogenersis of AD animal and its specific regulatory mechanisms are still not clear. MicroRNAs (miRNAs) are single-stranded noncoding short RNAs, which are important players in the processes of cell proliferation and differentiation. They also participate in certain diseases through regulating genes expression. In this project, we will measure the global miRNA expression by miRNA array and find differentially expressed miRNAs in AD transgenic mouse brain tissue under intervention of folic acid. The effects of folic acid on NSCs proliferation and differentiation will be detected in the same time. Bioinformatics tools and molecular biology techniques will be used to find candidate miRNAs which involved NSCs proliferation and differentiation and AD process. In order to study the regulation mechanism of candidate miRNAs in neurogenersis of AD animal, situ hybridization, real-time PCR and miRNA interference technology are used to confirm the expression of miRNAs and their target genes. The ultimate objective of this reseach are to explore the protective mechanism of folic acid on neurodegenerative disease and provide the scientific evidences for stimulating endogenous NSCs to treat AD.

阿尔茨海默病（AD）临床尚无特效治疗手段，寻求激活内源性神经干细胞（NSCs）治疗AD的方法是目前研究热点。前期研究发现叶酸能促进NSCs增殖分化，但其具体调控机制和对AD动物神经发生的作用尚不清楚。microRNA (miRNA) 是一类单链非编码小分子RNA，通过对基因表达的调控，在细胞增殖与分化及某些疾病发生过程中发挥重要作用。本研究拟运用miRNA芯片技术，筛选叶酸作用下AD转基因小鼠脑组织差异表达的miRNA，同时观察叶酸对AD小鼠NSCs增殖分化的影响，运用生物信息学手段和分子生物学技术寻找靶向NSCs增殖分化和AD相关基因的候选miRNA。利用原位杂交，实时定量PCR及miRNA干扰方法，在组织和细胞水平进行确证，深入研究候选miRNA对AD小鼠神经发生的作用和叶酸对其表达的影响，从而为探索利用内源性NSCs治疗AD和叶酸防治神经退行性疾病的机制提供科学依据。

阿尔茨海默病（AD）的发病机制复杂，至今尚无有效的治疗药物，研究发现叶酸与AD存在密切关系，AD患者miRNA的异常表达也是目前研究热点。为了探索叶酸通过miRNA途径对AD的作用机制，将APP/PS1双转基因小鼠分为AD模型组（AD+FN）、叶酸缺乏组（AD+FD）、叶酸低剂量组（AD+FL）、叶酸高剂量组（AD+FH），并设立阴性对照组（WT+FN）。Morris 水迷宫检测小鼠认知功能，免疫组化方法检测神经干细胞（NSCs）的增殖分化和淀粉样蛋白（Aβ）沉积。研究结果表明，叶酸缺乏可加重APP/PS1小鼠脑组织促进Aβ沉积，叶酸补充则降低Aβ沉积，但对APP/PS1小鼠认知功能的影响并不显著。未能发现叶酸对APP/PS1小鼠NSCs的明显影响。.miRNA芯片分析APP/PS1小鼠脑组织的miRNA表达谱，寻找差异miRNA，通过生物学信息技术预测靶向NSCs增殖分化和AD病程相关基因的miRNA分子，分析叶酸对候选miRNA表达的影响。结果显示，叶酸可调节APP/PS1小鼠脑组织miRNA表达，分析发现9个叶酸参与调控的、且可能与细胞生长和AD病程相关的差异miRNA，其中与APP（淀粉样前体蛋白），BACE1（淀粉蛋白前β位分解酶1）相关的miRNA有3个，即miR-106a-5p, miR-200b-3p和miR-339-5p。叶酸缺乏可明显促进小鼠脑组织APP和BACE1的蛋白表达，叶酸补充则抑制这两个基因的蛋白表达。体外培养N2a细胞，叶酸对转染APP的N2a和野生N2a细胞的这三个miRNA表达均产生影响。加入miRNA抑制剂后，靶基因表达增加，确定了差异miRNA和靶基因的关系。总之叶酸可以通过miRNA途径影响神经细胞和AD动物的Aβ沉积，对AD动物产生一定保护作用，本研究结果为阐明叶酸对AD等神经退行性疾病的预防与治疗作用提供了理论依据。