文档介绍：dsRNA阻断大鼠骨髓源性神经干细胞Hes5表达的实验研究
作者:王海燕1;徐如祥1;姜晓丹1;罗深秋2 (第一军医大学1珠江医院全军神经医学研究所,广东  广州 510282;2细胞生物学教研室学,广东  广州  510515)
摘要:目的  观察外源性短双链RNA(dsRNA)在转录后水平即mRNA水平降低基因表达的效率,并对其影响因素进行初步探讨。方法  将体外分离培养的大鼠骨髓基质细胞,通过由本实验室配制的特殊培养基诱导成神经干细胞,应用人工合成的dsRNA于不同浓度转染神经干细胞,Western bloting 检测dsRNA是否能阻断转录因子Hes5的表达,%锥虫蓝法测定各浓度组中培养细胞活力。结果  200~300 nmol/L浓度的dsRNA能特异有效地阻断Hes5的表达,而50~200 nmol/L浓度的dsRNA有利于干细胞存活。结论  dsRNA能在神经干细胞内启动RNA干扰作用,适宜浓度的dsRNA能在特异有效地阻断内源性基因的表达的同时,最大限度地保持细胞活力。
关键词:双链RNA;Hes5;神经干细胞,骨髓源性;RNA干涉;免疫印迹
中图分类号:Q189  文献标识码:A  文章编号:1000-2588(2004)01-0035-04
Blocking with double- stranded RNA of the expression of Hes5 in rat bone marrow-derived neural stem cells
WANG Hai-yan1; XU Ru-xiang1; JIANG Xiao-dan1; LUO Shen-qiu2
1Institute of Neuromedicine of PLA, Zhujiang Hospital, First Military Medical University, Guangzhou 510282,China; 2Department of Cellular Biology, First Military Medical University, Guangzhou 510515, China
Abstract: Objective  To examine the efficiency of exogenous small double-stranded RNA (dsRNA) in knocking down the gene expression at the post-transcription level, and investigate the factors that may influence the transfection. Method  The bone marrow stromal cells of SD rat were separated and cultured in vitro, followed by induction of the cells to evolve into neural stem cells using special culture medium prepared by our laboratory. Synthetic dsRNA was then transferred into the cells at varied concentrations, and the results were analyzed by Western blotting. Results  The concentrations ranging from 200 to 300 nmol/L were optimal for specifically blocking the expression of Hes5, whereas the suitable concentrations for the cell survival were between 50 and 200 nmol/L. Conclusion  dsRNA is capable of triggering RNA interference in neural stem cells, and at appropriate concentration, it may specifically and effectively knock down endogenous gene expression without sacrificing the viability of the cells.
Key words: double-stranded RNA; hairy and enhancer of spl