内源性siRNA对RAB5A和RABEX5表达的抑制作用  

Suppression Effect of Introgeneous siRNA on Expression of RAB5A and RABEX5

作　　者：张小爱[1] 侍庆[1] 吴华成[1] 曹文俊[1] 孙顺昌[1] 倪培华[1] 樊绮诗[1] 

机构地区：[1]上海交通大学医学院瑞金医院,上海200025

出　　处：《上海交通大学学报（医学版）》2006年第2期151-154,共4页Journal of Shanghai Jiao tong University:Medical Science

摘　　要：目的研究内源性的siRNA对RAB5A和RABEX5表达的抑制作用。方法利用siRNA表达载体pS ilencerTM2.1U6-neo对RAB5A和RABEX5基因分别构建3个特异的siRNA表达载体,并将表达载体分别转染人卵巢癌高转移细胞HO-8910PM。应用逆转录-聚合酶链反应和蛋白印迹检测转染前后HO-8910PM细胞中RAB5A和RABEX5的表达情况,并用荧光免疫细胞化学技术对干扰效率最好的载体进行验证。结果在构建的6个siRNA表达载体中,pS ilencer/siRAB5A-1和pS ilencer/siRAB5A-2对RAB5A表达的抑制率分别为90%和70%,pS ilencer/siRAB5A-3对RAB5A表达没有抑制作用。pS ilencer/siRABEX5-1、pS ilencer/siRABEX5-2和pS ilencer/siRABEX5-3对RABEX5表达的抑制率分别为30%、50%和90%。结论成功构建了对RAB5A和RABEX5表达具有抑制作用的siRNA表达载体,为进一步研究RAB5A和RABEX5的生物学功能提供了实验工具。Objective To investigate the suppressing effect of introgeneous siRNA on the expression of RAB5A and RABEXS. Methods Three siRNA expression vectors specific for RABSA and RABEX5 were constructed by using pSilencer^TM2. 1U6-neo, respectively, and the vectors for each gene were then transfected into high metastasis cell line HO-8910PM. The expression of RABSA and RABEX5 in HO-8910PM cells before and after transfection was detected by reverse transcription-polymerase chain reaction and Western blot. The vectors with the best inhibitory effect were confirmed by immunofluorescent cytochemistry analysis. Results The expression of RABSA was inhibited by transfecting with pSilencer/siRABSA-1 and pSilencer/siRABSA-2 with the efficiency of suppression of 90% and 70%. While pSilencer/siRABSA-3 failed to suppress the expression of RABSA. The inhibitory efficiency of pSilencer/siRABEXS-1 and pSilencer/siRABEXS-2 was about 30% and 50% , respectively, pSilencer/siRABEXS-3 had the highest efficiency on suppressing the RABEX5 expression （90%）. Conclusion The siRNA expression vectors of RABSA and RABEX5 were constructed successfully. It provided a powerful tool in studying the function of RABSA and RABEXS.

关 键 词：小干扰RNA RAB5A RABEX5 pSilencer载体 HO-8910PM细胞系 内源性siRNA 抑制作用 

分 类 号：Q75[生物学—分子生物学]