基于长链非编码RNA探讨调心方干预阿尔茨海默病前期病变作用机制

Alzheimer's disease (AD) is a kind of progressive neurodegenerative diseases. With higher incidence, the disease can be barely reversed when diagnosed. So, early intervention is undoubtedly more effective means to prevent the disease. Traditional Chinese medicine "Tiaoxin fang" for early intervention AD curative effect is distinct, but the mechanism is not clear. Some preliminary research shows that: long non-coding RNA (lncRNA) relative to maintain expression of Aβ protein and to maintain formation of AD. In our project, AD transgenic mouse was used as a model, lncRNA expression spectrum relative to AD development will be described firstly with lncRNA-microarray. Then, according to several lncRNA with high expression, special siRNA will be preparation and pack into slow virus particles. Then, the virus was injected into the hippocampus of model mouse which has not occur AD to make the lncRNA special silence. With immunohistochemistry, biochemistry, molecular biology and animal behavior analysis, the regulation effects of lncRNA in AD development will be discussed. At the same time, the AD model mouse will be treated with "Tiaoxin fang", to discusses whether the traditional Chinese medicine delay the onset of AD through the regulation of differences of the lncRNA expression. The aims of the project is to explain the expression of lncRNA related to AD, and whether "Tiaoxin fang" control the expression of lncRNA directly or indirectly, and effectively prevent the progression of AD. This project will provide new ideas of treatment and intervention to AD.

阿尔茨海默病(AD)危害极大，早期干预对其防治具有重要的意义。长链非编码RNA（lncRNA）参与维持Aβ的表达及AD的形成；而中医"调心方"是我国第一个被FDA批准用于AD治疗的中药，其深层次的作用机理有待阐明。本课题以AD转基因模型鼠作为研究对象，首先利用lncRNA芯片筛选出与AD发病相关的lncRNA表达谱。随后，针对若干个高表达的lncRNA制备siRNA慢病毒颗粒并注射于AD模型鼠（未发病）的海马部位，通过免疫组织化学、生化与分子生物学和动物行为学手段探讨上述lncRNA被特异性沉默后对于AD发病的调控作用。再者，分析lcnRNA在"调心方"干预鼠（未发病）与未干预之间的表达差异，研究该方药是否通过调控lncRNA的表达来延缓AD的发病。本课题旨在阐明中医"调心方"通过调控AD发病相关lncRNA的表达，有效阻止AD病程的发展，为AD前期病变的治疗和干预提供新的思路。

阿尔茨海默病（AD）的发生与细胞外β淀粉样蛋白(Aβ42/40)的沉积和细胞内tau蛋白过度磷酸化密切相关。长链非编码RNA（lncRNA）是一个非编码RNA家族的成员。虽然已经发现大量的lncRNA表达异常与哺乳动物神经系统发育和疾病有密切关系，但是其调节机制仍有待明确。已报到的β‑secretase‑1 (BACE1‑AS)是一个lncRNA分子，其在β‑分泌酶‑1（BACE1）升高的同时也升高表达，提示其在AD模型中正向调控BACE-1的表达和Aβ的聚集。本研究中，利用Aβ42寡聚肽处理SH-SY5Y细胞，建立AD细胞模型。借用该模型和siRNA技术，深入研究了lncRNA BACE1-AS表达与沉默过程中，BACE1加工APP的能力及Aβ42寡聚物生成的数量。MTT检测表明，外源性Aβ42寡聚肽可以有效抑制SH-SY5Y细胞增殖和诱导APP‑相关因子的表达和淀粉样斑块(SP)的形成。此外，qRT-PCR和Western blot分析显示，Aβ42处理组细胞内Aβ42/40的表达显著升高；而Ki‑67的表达显著下降。RPA和Northern blot印迹分析证实，外源性Aβ42不仅诱发了lncRNA BACE1-AS的表达，而且与BACE1 mRNA形成双链RNA从而增加了其稳定性，并保护了BACE1 mRNA不被RNase降解。此外，利用siRNA下调lncRNA BACE1‑AS在SH-SY5Y细胞内的表达可以导致BACE1加工Aβ前体APP能力的下降，并减弱SH-SY5Y细胞模型SP的形成。另一方面，lncRNA MEG3在Aβ1-42/1-40诱发神经元变性和损伤中的作用机制不明。我们借助Aβ42寡聚肽处理SH-SY5Y细胞，建立AD细胞模型。MTT实验表明，外源性的Aβ1-42可以显著抑制SH-SY5Y细胞的体外增殖能力。qRT-PCR和Western blot分析显示，在处理7天后，p53 mRNA和蛋白的表达在AD模型组显著升高，而MDM2与Ki-67的表达显著降低。此外，qRT-PCR和Northern blot分析证实，外源性Aβ1-42可以有效促进细胞内lncRNA MEG3的表达。当在AD细胞模型内过表达靶向沉默lncRNA Meg3的siRNA后，MDM2泛素化p53蛋白及其降解途径显著增强，而SH-SY5Y细胞的凋亡的显著变慢。