短干扰RNA对人β淀粉样前体蛋白裂解酶基因表达的影响

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）对BACE在神经母细胞瘤细胞中的表达的影响，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。结果限制性内切酶酶切和测序结果均表明成功构建了靶向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，该研究将为利用RNA干扰技术作为治疗AD的新方法提供重要的实验基础。

Specific Suppression of β-secretase Gene Expression by Short Interfering RNA in SK-N-SH Cells

OBJECTIVE: To test the effect of short interfering RNAs (siRNAs) of beta-site APP cleaving enzyme (BACE) on inhibiting the expression of BACE in mammalian cells. METHODS: The gene of EGFP, U6 promoter and beta-secretase targeting siRNA were cloned by PCR. The PCR products were inserted into the retrovirus plasmid pLXSN. The interfering vector was identified as pLXSN/ EGFP-U6-siBACE. The SK-N-SH cell line was produced, which can highly expressed BACE. The inhibitive effect of BACE siRNA on BACE expression was examined by fluoroscopy and immunohistochemistry tests. RESULTS: The interfering vector, pLXSN/EGFP-U6-siBACE, was constructed successfully. The BACE siRNA inhibited the expression of BACE in the SK-N-SH cell and reduced the production of Abeta. CONCLUSION: BACE siRNA inhibits the expression of BACE gene of mammalian, which has implications for RNA interference of Alzheimer's disease.