【摘要】 目的建立有活性的蛋白激酶C受体1(RACK1)过表达和低表达人脐静脉内皮细胞系(HUVEC),为进一步从分子水平研究RACK1在心律失常中的作用机制提供有效手段。方法扩增RACK1基因的全长c DNA序列,并插入p IRES2-EGFP获得过表达载体p IRES2-EGFP-RACK1;同时,设计合成3对短发夹结构的互补DNA序列和一对阴性对照序列,亚克隆至p Genesil-1得到相应的干扰载体;脂质体转染法将重组体及相应的空质粒分别转染至HUVEC细胞,经G418筛选抗性细胞克隆,qRT-PCR及Western blot鉴定RACK1 mRNA和蛋白的表达。结果RACK1真核表达载体和RNA干扰载体构建成功。重组体经脂质体法转染HUVEC 48 h后,经G418筛选3周得到细胞抗性克隆,qRT-PCR及Western blot结果证实了过表达载体和干扰载体能有效的增强和沉默HUVEC细胞中RACK1的表达。结论成功建立了RACK1过表达和低表达HUVEC细胞系。更多还原

【Abstract】 Objective To establish several human umbilical vein endothelial cell( HUVEC) strains with over-expression or low expression of receptor for activated C kinase 1( RACK1),which will provide an effective tool for future studying the function of RACK1 in arrhythmia. Methods The full-length c DNA sequence of RACK1 gene was amplified and inserted into p IRES2-EGFP. At the same time,designed and synthesised complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence,then subcloned into the plasmid p Genesil-1. The HUVEC cells were transfected with these plasmids and screened by using G418. And theexpression of RACK1 mRNA and protein in the cells were assayed by qRT-PCR and Western blot,respectively.Results RACK1 eukaryotic expression vector and siRNA expression vectors of RACK1 were constructed successfully. After a 48 h transfection of HUVEC cells with the recombinant vectors and G418 selection,the positive cell clones were obtained. qRT-PCR and Western blot showed that over-expression vector and interference vectors could effectively enhanced and knocked-down RACK1 expression in HUVEC strains. Conclusions HUVEC cell strains with over-expression and low expression of RACK1 have been successfully established.更多还原