摘要
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/ECFP-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。
Objective To test the effect of short interfering RNAs （siRNAs） of β-site APP cleaving enzyme （BACE） on inhibiting the expression of BACE in mammalian ceils. Methods The gene of EGFP, U6 promoter and β-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 Aβ. Conclusion BACE siRNA inhibits the expression of BACE gene of mammalian, which has implications for RNA interference of Alzheimer＇ s disease.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2006年第6期821-824,共4页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(批准号30300395,30400515)
教育部博士点基金(20030698011)资助~~
