摘要
目的构建核仁磷酸蛋白1(nucleophosmin1,NPM1)特异性的RNAi真核表达载体,观察对白血病K562细胞系NPM1表达的抑制作用。方法采用RT-PCR检测白血病细胞系(THP1和K562)和急性髓系白血病(AML)细胞NPM1mRNA水平。设计合成2条针对NPM1基因并具有小发夹结构的DNA序列,经退火形成互补双链,再克隆至载体pGenSil-1中构建含NPM1的RNAi重组体,经酶切及测序鉴定后通过脂质体转染K562细胞,同时设立pGenSil-1转染组和pGenSil-1/neg转染组。采用RT-PCR检测各组转染细胞中NPM1的mRNA水平。结果白血病细胞系和AML细胞均高表达NPM1 mRNA。针对人NPM1基因的RNAi重组体构建成功,并能够稳定转染K562细胞,导致白血病细胞NPM1mR-NA相对表达量下降64%,而pGenSil-1转染组和pGenSil-1/neg转染组未能下调NPM1表达。结论白血病细胞高表达NPM1基因,其mRNA表达水平能够被靶向NPM1的RNAi重组体特异性地下调。
Objective To construct eukaryotic expression vector of RNAi specific to nucleophosminl （ NPM1 ） and investigate the effect of recombinant plasmid on NPM1 expression in K562 cells. Methods The expression level of NPM1 mRNA was detected by RT-PCR in leukemia cell lines （THP1 and K562）, bone marrow or blood samples from 5 patients with acute myelogenous leukemia （AML） and from 5 healthy subjects. A short hairpin RNA targeting NPM1 was synthesized and inserted into the plasmid pGenSil-1. After the recombinant plasmid was identified, the correct plasmid was transfected into K562 cells, then the expression level of NPM1 mRNA was detected by RT-PCR. Results NPM1 mRNA levels were higher in two leukemia cell lines and all AML samples than those in normal cells. The RNAi recombinant plasmid was constructed successfully. The NPM1 mRNA levels decreased by 64% in K562 cells after the recombinant plasmid transfection, but those were not inhibited in K562 cells transfected with empty plasmid and untransfected K562 cells. Conclusion NPM1 mRNA is overexpressed in leukemia cells. The RNAi recombinant targeting NPM1 suppresses the expression of NPM1 mRNA.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第10期938-941,共4页
Journal of Third Military Medical University
基金
重庆市教委科学技术研究基金(KJ050309)~~
