慢病毒介导的GSK-3β基因稳定沉默表达的SH-SY5Y细胞系建立

发布时间：2019-04-17 06:12

【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P0.01)。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。
[Abstract]:Aim to construct RNA interference lentivirus vector targeting glycogen synthesis kinase 3 尾 (GSK-3 尾) gene and establish a cell line model of human neuroblastoma cell line (SH-SY5Y) whose expression of GSK-3 尾 gene is silenced. Methods according to the GSK-3 尾 mRNA sequence, the short hairpin RNA (shRNA) sequence of the target gene was designed and synthesized, and the lentivirus expression vector was constructed. The recombinant lentiviral vector was co-transfected with the packaging vector to 293T cells by restriction endonuclease digestion and sequencing. SH-SY5Y cells were infected with purified and concentrated virion. The transfected cells were divided into lentivirus interference group (experimental group), negative control group (Lenti-NC group) and blank control group (Blank group) by green fluorescence tracer. The expression levels of GSK-3 尾 mRNA and protein were detected by qRT-PCR and Western blot methods. Results shRNA lentivirus targeting GSK-3 尾 was successfully constructed. Recombinant lentivirus was transfected into human SH-SY5Y cells. After 72 hours, the transfection efficiency was more than 90%. Compared with the control group, the target gene changes showed that the recombinant lentivirus was transfected into SH-SY5Y cells. The expression of GSK-3 尾 mRNA and protein in the experimental group was significantly lower than that in the control group (P0.01). Conclusion the lentivirus-mediated stable silencing expression of GSK-3 尾 gene in SH-SY5Y cell model can be established effectively, which provides an experimental tool for evaluating the drug or behavioral therapy of AD after GSK-3 尾 silencing in vitro.
【作者单位】：山东大学附属济南市中心医院神经内科;济南市第四人民医院内分泌科;
【基金】：国家自然科学基金资助项目(No:30970989)
【分类号】：R749.16

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