短干扰RNA特异性抑制哺乳动物β淀粉样前体蛋白裂解酶基因表达

目的：探讨β淀粉样前体蛋白裂解酶（β-site APP cleaving enzyme，BACE）的短干扰RNA（short interfering RNAs，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）、U6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-U6-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-U6-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-U6-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。

Specific suppression of β-secretase gene expression by short interfering RNA in mammalian cells

OBJECTIVE: To investigate whether short interfering RNAs(siRNAs) of beta-site APP cleaving enzyme (BACE) can inhibit the expression of BACE in mammalian cells. METHODS: The gene of EGFP, U6 promoter and beta-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. RESULT: 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 beta was reduced. CONCLUSION: BACE siRNA can inhibit the expression of BACE gene of mammalian cells.