siCBP慢病毒降低大鼠原代海马神经元乙酰化组蛋白表达  

Acetylated Histone Expressions of the Primary Hippocampal Neurons in Rats Reduced by siCBP Lentivirus

作　　者：侯娜丽 吴小凤[1] 任兰[1] 郭敏[1] 毕杨[1] 刘友学[1] 陈洁[1] 黄鸿眉[1] 李廷玉[1] 

机构地区：[1]重庆医科大学附属儿童医院儿童营养研究中心,儿童发育疾病研究教育部重点实验室,重庆市认知与学习记忆障碍重点实验室,重庆400014

出　　处：《生物医学工程学杂志》2015年第4期838-846,共9页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(81161120498,81100476);高等学校博士学科点专项科研基金资助项目(20115503110003)

摘　　要：本研究目的为构建携带针对大鼠环磷酸腺苷反应元件结合因子的结合蛋白(CBP)基因的siRNA重组慢病毒,感染原代海马神经元并检测其对乙酰化组蛋白表达的抑制作用。本文构建了4种siCBP,并检测其对神经元中CBP表达的抑制作用,选择效果最好的一种检测神经元中HAT活性及乙酰化组蛋白Ac-H3、Ac-H4的表达。酶切及测序鉴定证实siCBP正确克隆至慢病毒质粒中,4种siCBP感染原代海马神经元后CBP mRNA和蛋白表达有不同程度降低,效果最佳的GR806对神经元的HAT活性及Ac-H3、Ac-H4表达均有显著抑制作用。本研究为后续应用siCBP阐明CBP依赖的组蛋白乙酰化对海马区学习记忆功能调节的相关分子机制提供了实验工具。This study aims to construct the recombinant lentivirus vector containing specific small interfering RNA （siRNA） targeting rat CREB binding protein（CBP）gene and to identify its function of inhibiting the expressions of acetylated histone in primarily cultured hippocampal neurons. Firstly, we constructed four kinds of recombinant lentivirus siCBP. And then we used them to infect the primarily cultured hippocampal neurons, and performed realtime PCR, western blot respectively to detect the expressions of CBP. Afterwards, the most effective lentivirus siCBP was used to infect the primarily cultured hippocampal neurons, and then the HAT activity and protein expres- sions of acetylated histone Ae-H3, Ac-H4 of the neurons were examined. By using PCR, endonuclease cutting and gene sequencing, we confirmed that the target genes were correctly cloned in lentivirus vector. Besides, CBP mRNA and protein expressions in neurons were found to be with varying degrees of decreases after infections of the four kinds of lentivirus siCBP. Furthermore, the representative and most effective lentivirus GR806 could effectively in- hibit the HAT activity and the protein expressions of Ac H3, Ac-H4 in neurons. It provides the experimental basis for the subsequent application of siCBP to clarify the effects and corresponding molecular mechanism of the CBP-de pendent histone acetylation on learning and memory function in hippocampus.

关 键 词：环磷酸腺苷反应元件结合因子的结合蛋白 慢病毒载体 siRNA 海马神经元 组蛋白乙酰化 

分 类 号：R741[医药卫生—神经病学与精神病学]