【摘要】 目的:探讨β淀粉样前体蛋白裂解酶(-βsite APP cleav ing enzym e,BACE)的短干扰RNA(short interfering RNA s,siRNA)是否能抑制BACE在哺乳动物细胞中的表达,为阿尔茨海默病(AD)的治疗提供新的手段。方法:采用PCR分别扩增增强型绿色荧光蛋白(EGFP)、U 6启动子和靶向BACE的特异性小干扰RNA(siBACE),随后将相应的序列片段克隆入真核表达载体pLXSN,通过限制性内切酶和测序对该重组表达载体pLXSN/EGFP-U 6-siBACE进行鉴定;制备稳定高表达BACE基因的神经母细胞瘤SK-N-SH细胞株,用荧光显微镜和免疫组织化学的方法观察siRNA对BACE表达的影响。结果:成功构建了靶向BACE的短干扰RNA的逆转录病毒载体pLXSN/EGFP-U 6-siBACE,并能特异性地抑制BACE在神经母细胞瘤SK-N-SH细胞株中的表达。结论:成功构建了针对BACE的短干扰RNA的逆转录病毒表达载体pLXSN/EGFP-U 6-siBACE,并能有效抑制哺乳动物BACE基因的表达,为利用RNA干扰技术作为治疗AD的新方法提供了重要的实验基础。更多还原

【Abstract】 Objective: To investigate whether short interfering RNAs(siRNAs) of β-site APP cleaving enzyme(BACE) can inhibit the expression of BACE in mammalian cells.Methods: The gene of EGFP,U6 promoter and β-secretase targeting siRNA were cloned by PCR,respectively.The PCR products were inserted into plasmid pLXSN.The interfering vector pLXSN/EGFP-U6-siBACE was transferred into SK-N-SH cells to express BACE.The inhibition effect of BACE siRNA on BACE expression was investigated by fluoroscopy and immunohistochemistry method.Results: The interfering vector pLXSN/EGFP-U6-siBACE was constructed successfully.The BACE siRNA inhibited the expression of BACE in the SK-N-SH cells specifically and effectively,and the production of A β was reduced.Conclusion: BACE siRNA can inhibit the expression of BACE gene of mammalian cells.更多还原