β-arrestin2抗基因RNA慢病毒载体体外基因沉默效应研究

摘要

目的:探讨β-arrestin2抗基因RNA慢病毒载体在体外工具细胞中的基因沉默效应及其携带的用于活体核磁共振成像监测的铁蛋白报告基因的表达.方法:用构建的β-arrestin2抗基因RNA慢病毒感染NG108-15细胞,实时荧光定量PCR和Western Blot方法检测β-arrestin2抗基因RNA慢病毒对工具细胞的β-arrestin2 mRNA和蛋白表达的下调作用,以及该病毒所携带的用于活体核磁共振成像监测的铁蛋白报告基因的表达.结果:构建的β-arrestin2抗基因RNA慢病毒能显著降低工具细胞中β-arrestin2 mRNA和蛋白的表达,与对照组相比实时荧光定量PCR和Western Blot方法检测的沉默效率分别为79%和82%(P<0.05).感染病毒组的NG108-15细胞的铁蛋白表达量显著升高,是对照组细胞的2.29倍(P<0.05).结论:构建的β-arrestin2抗基因RNA慢病毒在工具细胞中对β-arrestin2基因具有显著的沉默效应,该病毒携带的铁蛋白报告基因在工具细胞中表达.成功验证了β-arrestin2抗基因RNA慢病毒在体外的基因沉默效应和铁蛋白报告基因的表达,为该病毒的在体研究奠定了基础.%Objective: To study gene silencing effect of β-arrestin2 antigene RNA lentivirus in vitro and its expression of reporter gene ferritin, which can be further used for magnetic resonance imaging (MRI)in vivo. Methods: NG108-15 cells were infected by recombinant β-arrestin2 antigene RNA lentivirus vector. Real-time quantitative PCR and Western Blot were utilized to detect the down-regulation of β-arrestin 2 in the cells infected by the lentivirus vector at the mRNA and protein level, respectively. The expression of ferritin, which can be monitored by MRI in vivo, was also determined. Results: The β-arrestin2 expression was significantly decreased by the resultant recombinant lentivirus vector, and the gene silencing efficiency was 79％ in the RNA level and 82％ in the protein level (P ＜0.05). However, the expression of ferritin in NG108-15 cells infected by β-arrestin2 antigene RNA lentivirus vector was significantly increased 2.29 times than that in naive group ( P ＜ 0.05 ). Conclusions: The β-arrestin2 expression was significantly decreased by the constructed lentivirus vector in vitro, while the expression of ferritin was increased profoundly. The gene silencing effect of β-arrestin2 antigene RNA lentivirus vector and its expression of ferritin in vitro were successfully identified, consequently foundation for the latter research in vivo was laid.