CAJ | 학술논문

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.
목적 탐토β정분양전체단백렬해매(β-site APP cleaving enzyme,BACE)적단간우RNA(short interfering RNAs,siRNA)대BACE재신경모세포류세포중적표체적영향,위아이자해묵병(AD)적치료제공신적수단.방법 채용PCR분별확증증강형록색형광단백(EGFP)、U6계동자화파향BACE적특이성소간우RNA(siBACE),수후장상응적서렬편단극륭입진핵표체재체pLXSN,통과한제성내절매화측서대해중조표체재체pLXSN/EGFP-U6-siBACE진행감정;제비은정고표체BACE기인적신경모세포류SK-N-SH세포주,용형광현미경화면역조직화학적방법관찰siRNA대BACE표체적영향.결과 한제성내절매매절화측서결과균표명성공구건료파향BACE적단간우RNA적역전록병독재체pLXSN/EGFP-U6-siBACE;병능특이성지억제BACE재신경모세포류SK-N-SH세포주중적표체.결론 성공구건료침대BACE적단간우RNA적역전록병독표체재체pLXSN/EGFP-U6-siBACE,병능유효억제포유동물BACE기인적표체,해연구장위이용RNA간우기술작위치료AD적신방법제공중요적실험기출.
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 cells. 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.