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首页> 外文期刊>Acta Pharmacologica Sinica >Down-regulation amyloid β-protein 42 production by interfering with transcript of presenilin 1 gene with siRNA
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Down-regulation amyloid β-protein 42 production by interfering with transcript of presenilin 1 gene with siRNA

机译：siRNA干扰早老素1基因的转录下调淀粉样β蛋白42的产生

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AIM: To investigate the pathogenesis of Aβ_(42) yielding and new drug targets as well as the possibility of RNA interference (RNAi) technique for treatment of Alzheimer disease (AD). METHODS: 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 G_(418). Transcripts of PS1 gene in CHO/PS1/APP were measured by RT-PCR, the contents of PS1 peptide and Aβ_(42) production inside CHO/PS1/APP cells were examined through Western blot and the Aβ_(42) change of secretion by CHO/PS1/APP was determined with ELISA. RESULTS: 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 Aβ_(42) production in CHO/PS1/APP cells were both reduced after siRNA interfere for 3 d. Aβ_(42) 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 Aβ_(42) with RNAi action. CONCLUSION: PS1 is essential for γ-secretase activity. Inhibition of the PS 1 can decrease the levels of Aβ_(42). Some sites of PS 1 mRNA, for example, the site 3, may serve as a new drug target and RNAi probably can be used for treatment of AD.
机译：目的：探讨产生Aβ_（42）和新药靶标的发病机制，以及RNA干扰（RNAi）技术治疗阿尔茨海默病（AD）的可能性。方法：从NCBI网站获得人AD早老素1（PS1）cDNA序列。通过Ambion公司的在线设计，在PS1 cDNA中选择了三个RNAi作用位点和一个错义控制位点。为了确认这些位点的特异性，我们对IMAGE EST库进行了BLAST搜索。相应的双链DNA被用于构建pSilencer 3.1-H1质粒，该质粒可以转录小干扰RNA（siRNA）。然后，用SuperFect转染试剂将pSliencer 3.1-H1质粒转染到CHO / PS1 / APP细胞中。已经用AD的突变PS1和APP基因转染了细胞。使用G_（418）筛选出所有带有pSliencer 3.1-H1质粒的CHO / PS1 / APP细胞。通过RT-PCR检测CHO / PS1 / APP中PS1基因的转录本，通过Western blot检测CHO / PS1 / APP细胞内PS1肽的含量和Aβ_（42）的产生，并通过免疫印迹法检测分泌的Aβ_（42）的变化。 CHO / PS1 / APP用ELISA测定。结果：2 d后，RNAi抑制了PS1 mRNA的位点3。随着时间的推移效果更加明显。 siRNA干扰3 d后，CHO / PS1 / APP细胞中对应于PS1基因的肽和Aβ_（42）的产生均降低。 CHO / PS1 / APP细胞的Aβ_（42）分泌在第3天开始减少，并在第5天达到最大。在PS1基因的转录本与Aβ_（42）的产生之间存在时间依赖性。 RNAi作用。结论：PS1是γ分泌酶活性必不可少的。 PS 1的抑制可降低Aβ_（42）的水平。 PS 1 mRNA的某些位点（例如位点3）可以用作新药靶标，RNAi可能可以用于治疗AD。

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