siRNA表达载体对293T细胞neuritin表达的抑制作用

Inhibiting effect of siRNA expression vector on neuritin in 293T cells

目的观察siRNA表达载体对293T细胞神经生长因子neuritin的抑制作用,为进一步研究neuritin基因的功能及作用机制奠定基础。方法通过RT-PCR技术筛选neuritin高表达细胞株;利用分子克隆技术,采用两步法构建PBS/U6-neuritin siRNA(1～3)表达载体,脂质体法转染293T细胞,稳定转染后采用Western-blot技术进行干扰效率的筛选鉴定。结果 293T细胞与L-02细胞的RT-PCR产物为500bp左右可见特异性条带,选择293T细胞作为实验细胞株,测序结果显示经2次构建后PBS/U6-neuritin siRNA表达载体在338bp处出现发夹结构,说明构建成功。转染72h后Western-blot技术检测PBS/U6-neuritin siRNA-(1～3)在不同程度上均可使293T细胞内源性Neuritin蛋白表达下调。结论 PBS/U6-neuritin siRNA(1～3)表达可以有效抑制293T细胞系Neuritin蛋白的表达。

Objective To investigate the inhibitory effects of siRNA expression of Neurotrophic-factor basis for further research about the biological fuction and action mechanism of Neuritin.Mothods The high expression cell line of neuritin was selected by reverse transcription polymerase chain reaction（RT-PCR）;PBS/U6-neuritin siRNA（1~3） expression vectors were constructed by two stepts with molucular cloning technique.The recombinants were transfected into 293T cells with liposome.The expression of neuritin was checked by Western-blot.Results The 293T cells and L-02 cells had specific PCR product in 500 bp,so we chose 293T cell line to be experiment cell line.PBS/U6-neuritin siRNA expression vectors produced specific hairpin siRNA in 338 bp by sequence analysis,which proved successful Instruction.Transfection with PBS/U6-neuritin siRNA（1~3） expression vectors could inhibit Neuritin expression in 293T cell line by Western-blot in 72 th hour.Conclusion PBS/U6-Neuritin siRNA（1~3） expression vectors were successfully extablished that can effectively inhibit the expression of Neuritin in 293T cells.