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目的探讨AIF基因短发夹RNA(shRNA)表达质粒对神经母细胞瘤细胞(SHSY5YAIF)基因的干扰作用。方法根据siRNA设计原则,构建靶向AIF基因的短发夹RNA干扰质粒(pGPU6/GFP/AIF),使用脂质体法转染人神经母细胞瘤细胞,通过RT-PCR和Wester blot印迹检测神经母细胞瘤细胞AIF基因mRNA和蛋白表达水平。结果 pGPU6/GFP/AIF重组质粒经限制性内切酶酶切及测序证明基因插入正确。质粒成功转染SHSY5Y细胞后,该细胞的AIFmRNA和蛋白表达明显下降(P<0.05)。结论成功构建靶向AIF基因的shRNA表达载体转染神经母细胞瘤细胞,有效抑制AIFmRNA和蛋白表达,为AIF基因靶向治疗提供前期的实验依据。
Objective To investigate the interference of AIF gene short hairpin RNA (shRNA) expression plasmid on neuroblastoma cells (SHSY5YAIF). Methods According to the principle of siRNA design, short interfering RNA interference plasmid (pGPU6 / GFP / AIF) targeting AIF gene was constructed and transfected into human neuroblastoma cells by lipofectamine. The neurons were detected by RT-PCR and Wester blot AIF mRNA and protein expression levels in blast cells. Results The pGPU6 / GFP / AIF recombinant plasmid was verified by restriction enzyme digestion and sequencing. The expression of AIF mRNA and protein in SHSY5Y cells was significantly decreased after transfected with plasmids (P <0.05). Conclusion The shRNA expression vector targeting AIF gene was successfully transfected into neuroblastoma cells, which can effectively inhibit the expression of AIF mRNA and protein and provide the experimental basis for the targeted therapy of AIF gene.