Complexin-1/2过表达重组载体对阿尔茨海默病小鼠记忆损伤的实验研究  

Construction of adeno-associated virus over- expressing vector of complexin-1/2 gene and overexpres-sion of complexin-1/2 on spatial memory impairment in mice of Alzheimer's disease

作　　者：张艳玲[1,2] 刘建军[2] 许华[1] 杨细飞[2] 

机构地区：[1]暨南大学药学院,广州510632 [2]深圳市疾病预防控制中心,深圳市现代毒理学重点实验室,深圳518055

出　　处：《中国临床解剖学杂志》2016年第2期191-197,共7页Chinese Journal of Clinical Anatomy

基　　金：广东省自然科学基金(2014A030313715);深圳市科技研发基金基础研究项目(JCYJ20130329103949650)

摘　　要：目的构建Complexin-1/2(CPLX-1/2)基因的重组腺相关病毒过表达载体p AAV-CPLX-1/2并研究其在阿尔茨海默病记忆损伤中的作用。方法将用Oligo 7软件对Genbank上小鼠CPLX-1/2m RNA的CDS两端序列进行分析,合成CPLX-1/2基因,构建过表达载体并转化至大肠杆菌Top10;选取正确的克隆进行PCR和测序鉴定。重组腺相关病毒载体测序正确后,转染AAV293细胞,包装腺相关病毒。将重组腺相关病毒经脑立体定位注射到三转基因阿尔茨海默病(3XTg-AD)小鼠海马CA3区,经免疫组化和Western-Blot检测CPLX-1/2蛋白表达情况,水迷宫检测CPLX-1/2过表达对3XTg-AD小鼠记忆的影响。结果 PCR及测序结果表明成功构建了含小鼠CPLX-1/2基因的重组腺相关病毒表达载体。CPLX-1/2在海马CA3表达显著增加。CPLX-1/2过表达显著改善3XTg-AD小鼠的记忆损伤。结论本研究成功构建了CPLX-1/2重组腺相关病毒过表达载体并在整体动物水平成功高效表达;CPLX-1/2过表达可显著改善3XTg-AD小鼠空间记忆的损伤。为在整体动物水平深入研究CPLX-1/2的功能提供了新的手段;研究结果提示CPLX-1/2可能作为AD记忆损伤治疗的关键靶点。Objective To construct recombinant adeno- associated virus over- expressing vector of complexin- 1/2（CPLX- 1/2） and investigate the effects of overexpression of CPLX- 1/2 on spatial memory impairment in Alzheimer＇s Disease. Methods The CPLX- 1/2 gene sequences of mice reported in Genbank were analyzed by Oligo software, and the CPLX- 1/2 gene was synthesized. The overexpressing vector was constructed and transfected into Escherichia coli TOP 10. Purified plasmids from the positive clones was confirmed by PCR and sequencing. The AAV293 cells were transfected by the recombinant adenoassociated virus vector to achieve the adeno- associated virus packaging production. The recombinant adenoassociated virus vector was injected into hippocampus of 3XTg- AD mice using a stereotaxic instrument guided by a computer and a motorized nanoinjector. The expression of CPLX- 1/2 in hippocampus was detected by Western-blot and immunofluorescence; Morris water maze was performed to measure the change of spatial memory impairment in 3XTg- AD mice. Results The PCR and sequencing confirmed that the recombinant adeno-associated virus vector was successfully constructed. Injection of the overexpessing vector led to significantly increased expression of CPLX-1/2 in hippocampus, and significantly improved the spatial memory impairment in 3XTg-AD mice. Conclusion The adeno-associated virus overexpressing vector of CPLX- 1/2 gene was successfully constructed, and highly expressed in hippocampus of 3XTg- AD mice. The expression of CPLX- 1/2 gene in hippocampus significantly improved spatial memory impairment in 3XTgAD mice. This study provides a new approach to further study the function of CPLX- 1/2 at in vivo levels.These findings suggest that CPLX-1/2 may serve as key therapeutic targets for AD.

关 键 词：阿尔茨海默病 Complexin-1/2 重组腺相关病毒载体 基因过表达 

分 类 号：R742[医药卫生—神经病学与精神病学]