Luo Huan-min, Deng Hui, Xiao Fei, Gao Qin, Weng Wen, Zhang Pei-fen, Li Xiao-guang
Neuropharmacological Research Laboratory, College of Pharmacy, Ji-nan University, Guangzhou 510632, China.
Acta Pharmacol Sin. 2004 Dec;25(12):1613-8.
To investigate the pathogenesis of Abeta42 yielding and new drug targets as well as the possibility of RNA interference (RNAi) technique for treatment of Alzheimer disease (AD).
Human AD presenilin 1 (PS1) cDNA sequence was obtained from NCBI website. The three sites of RNAi action and one missense control site were selected in PS1 cDNA through online design of Ambion company. To confirm specificity of these sites, we conducted a BLAST search of the IMAGE EST library. The corresponding double-stranded DNA was used to construct pSilencer 3.1-H1 plasmid, which could transcribe small interference RNA (siRNA). Then, the pSliencer 3.1-H1 plasmids were transfected into CHO/PS1/APP cells with SuperFect transfection reagent. The cells have been transfected with the mutant PS1 and APP gene of AD. All the CHO/PS1/APP cells with pSliencer 3.1-H1 plasmids were screened out using G418. Transcripts of PS1 gene in CHO/PS1/APP were measured by RT-PCR, the contents of PS1 peptide and Abeta42 production inside CHO/PS1/APP cells were examined through Western blot and the Abeta42 change of secretion by CHO/PS1/APP was determined with ELISA.
The site 3 of PS1 mRNA was inhibited by RNAi after 2 d. The effect was more obvious with the time. The peptide corresponding to PS1 gene and Abeta42 production in CHO/PS1/APP cells were both reduced after siRNA interfere for 3 d. Abeta42 secretion by CHO/PS1/APP cells began to reduce on d 3, and reached the most significance on d 5. There was a time-dependent relationship between the transcript of PS1 gene and the production of Abeta42 with RNAi action.
PS1 is essential for g-secretase activity. Inhibition of the PS1 can decrease the levels of Abeta42. Some sites of PS1 mRNA, for example, the site 3, may serve as a new drug target and RNAi probably can be used for treatment of AD.
探讨β淀粉样蛋白42（Aβ42）产生的发病机制、新的药物靶点以及RNA干扰（RNAi）技术治疗阿尔茨海默病（AD）的可能性。
从NCBI网站获取人AD早老素1（PS1）的cDNA序列。通过Ambion公司的在线设计，在PS1 cDNA中选择3个RNAi作用位点和1个错义对照位点。为确认这些位点的特异性，我们对IMAGE EST文库进行了BLAST搜索。用相应的双链DNA构建可转录小干扰RNA（siRNA）的pSilencer 3.1-H1质粒。然后，用SuperFect转染试剂将pSliencer 3.1-H1质粒转染到CHO/PS1/APP细胞中。这些细胞已转染了AD的突变PS1和APP基因。用G418筛选出所有转染了pSliencer 3.1-H1质粒的CHO/PS1/APP细胞。用逆转录-聚合酶链反应（RT-PCR）检测CHO/PS1/APP中PS1基因的转录本，通过蛋白质印迹法检测CHO/PS1/APP细胞内PS1肽的含量和Aβ42的产生，并用酶联免疫吸附测定（ELISA）法测定CHO/PS1/APP分泌的Aβ42的变化。
RNAi作用2 d后，PS1 mRNA的位点3受到抑制。随着时间的推移，这种作用更明显。小干扰RNA干扰3 d后，CHO/PS1/APP细胞中与PS1基因对应的肽和Aβ42的产生均减少。CHO/PS1/APP细胞分泌的Aβ42在第3天开始减少，在第5天达到最显著水平。PS1基因转录本与RNAi作用下Aβ42产生之间存在时间依赖性关系。
PS1对γ-分泌酶活性至关重要。抑制PS1可降低Aβ42水平。PS1 mRNA的某些位点，如位点3，可能成为新的药物靶点，RNAi可能可用于治疗AD。
