慢病毒介导的GSK-3β基因稳定沉默表达的SH-SY5Y细胞系建立  

Construction of human SH-SY5Y cell line stably transfected with GSK-3β gene silencing expression by lentivirusmediated RNA interference

作　　者：边红[1] 边卫[2] 孙金波[1] 李严霜[1] 陈雯[1] 

机构地区：[1]山东大学附属济南市中心医院神经内科,山东济南250013 [2]济南市第四人民医院内分泌科,山东济南250031

出　　处：《中国现代医学杂志》2014年第6期14-18,共5页China Journal of Modern Medicine

基　　金：国家自然科学基金资助项目(No:30970989)

摘　　要：目的构建靶向糖原合成激酶3β(GSK-3β)基因的RNA干扰慢病毒载体,建立其对GSK-3β基因沉默表达的人神经母细胞瘤细胞(SH-SY5Y)的细胞系模型。方法根据GSK-3βmRNA序列设计合成靶向目的基因的短发夹状RNA(shRNA)序列并构建慢病毒表达载体,经酶切和测序鉴定,将慢病毒重组载体与包装载体共转染293T细胞,获得纯化浓缩病毒液,侵染SH-SY5Y细胞,采用绿色荧光示踪观察细胞的转染情况,设为慢病毒干扰组(实验组)、阴性对照组(Lenti-NC组)、空白对照组(Blank组),利用qRT-PCR和Western blot法检测GSK-3βmRNA和蛋白表达水平。结果成功构建了靶向GSK-3β的shRNA慢病毒,重组慢病毒转染人SH-SY5Y细胞,72 h后观察转染效率达90%以上,与对照组比较,靶基因变化结果显示,实验组中GSK-3βmRNA和蛋白的表达均显著降低(均P<0.01)。结论有效建立慢病毒介导的GSK-3β基因稳定沉默表达的SH-SY5Y细胞模型,为体外评价研究GSK-3β沉默后药物或行为治疗AD提供试验工具细胞模型。[Objective] To construct lentiviral vector of RNA interference targeting GSK-3β gene and establish neuroblastoma tumor cell （SH-SY5Y） line model with GSK-3β gene silencing expression. [ Methods ] According to GSK-3β mRNA, we designed and synthesized the target gene short hairpin RNA （shRNA） sequences and construct- ed a lentiviral vector. The recombinant lentiviral vectors were confirmed by restriction enzymes, PCR and sequencing. 293T cells were co-transfected with lentiviral vector and packaging system. SH-SYSY cells were divided into experimental group （infected with the GSK-3β shRNA）, Lenti-NC group （infected with control-shRNA） and blank control group （without transfection）. Fluorescence microscope was used to observe the transfection efficiency. Real time PCR and Western blot were used to examine the expression of GSK-3β mRNA and protein in SH-SY5Y cells. [Results] The recombinant lentivirus vector of GSK-3β was successfully constructed and the lentivirns can infect cells more than 90%. Compared with the Lenti-NC group and blank control group, the expression of GSK-3β mR- NA and protein was significantly down-regulated in experimental group. [Conclusions] Human SH-SY5Y cell model with GSK-3β gene silencing expression by lentivirus-mediated RNA interference were effectively established,which could provide a good test cell model for in vitro evaluation for drug or behavioral treatment of AD after GSK- 3β gene silence.

关 键 词：阿尔茨海默病 慢病毒属 RNA干扰 糖原合成激酶-3β 

分 类 号：R749.1[医药卫生—神经病学与精神病学]