MicroRNA在建立DJ-1基因敲减细胞模型中的应用  

Application of two types of RNA to develope DJ-1 knockdown cell model

作　　者：赵莎莎[1] 鲁玲玲[1] 高华[1] 吕瓅[1] 赵焕英[1] 杨慧[1] 

机构地区：[1]首都医科大学神经生物学系北京神经科学研究所北京市神经再生修复研究重点实验室神经变性病教育部重点实验室,北京100069

出　　处：《基础医学与临床》2011年第5期515-519,共5页Basic and Clinical Medicine

基　　金：国家"973"计划(2006CB500706;2011CB504103);国家"863"计划(2006AA02A408);北京市自然科学基金(5102007;5102012;7082011);国家自然科学基金(30670655;30950003;30970940和30700199);北京市优秀人才培养项目(20081d0501800210);北京市高校人才强教创新团队计划(PHR200907113)

摘　　要：目的与传统的siRNA方法建立基因敲减模型不同,探讨合成microRNA（miRNA）在DJ-1基因敲减细胞模型中的应用。方法构建针对DJ-1基因的合成miRNA载体,合成DJ-1基因的siRNA干扰片段。在脂质体介导下,将上述两种小干扰RNA载体或片段分别转染MN9D细胞系,应用real-time PCR和Western blot检测目的基因DJ-1的mRNA和蛋白表达。结果与对照组相比,转染合成microRNA的MN9D细胞中,DJ-1的mRNA水平下调90%（P〈0.05）,蛋白水平下调70%~85%（P〈0.05）;而转染siRNA的MN9D细胞中,DJ-1的mRNA水平下调50%~70%（P〈0.05）,蛋白水平下调20%~50%（P〈0.05）。结论 microRNA与siRNA均可作为基因敲减的重要研究手段。与传统的siRNA相比,合成microRNA对目的DJ-1的干扰效率更为显著。Objective To develope gene knockdown cell model with artificial microRNA in setting up gene knockdown cell model.Methods We constructed vectors,and prepared siRNA fragments targeting on DJ-1.Then we transciently transfected the artificial miRNA and siRNA into MN9D cells by lipofectamine2000 reagent,the mRNA and protein expression level of DJ-1 gene were detected by RT-PCR and Western blot.Results Compared with control group,DJ-1 expression level was significantly decreased in both artificial miRNA and siRNA groups.DJ-1 was knockdowned and DJ-1 was decreased 90%（P0.05）at mRNA expression level,and decreased 70%~85%（P0.05） at protein level in MN9D cells transfected with the artificial miRNA.While DJ-1 was decreased by 50%~70%（P0.05）at mRNA level,and decreased by 20%~50%（P0.05）at protein level in MN9D cells transfected with siRNA.Comparing with siRNA,miRNA was more effective in silencing DJ-1.Conclusion The artificial miRNA and siRNA are both effective in silencing gene.miRNA has more significant function in knockingdown DJ-1 than siRNA.

关 键 词：DJ-1 MICRORNA SIRNA RNA干扰 

分 类 号：R742.5[医药卫生—神经病学与精神病学]