CAJ | 학술논문

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cDNA序列，并插入pIRES2-EGFP获得过表达载体pIRES2-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pGenesil-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。
목적：건립유활성적단백격매C수체1（RACK1）과표체화저표체인제정맥내피세포계（HUVEC），위진일보종분자수평연구RACK1재심률실상중적작용궤제제공유효수단。방법확증RACK1기인적전장cDNA서렬，병삽입pIRES2-EGFP획득과표체재체pIRES2-EGFP-RACK1；동시，설계합성3대단발협결구적호보DNA서렬화일대음성대조서렬，아극륭지pGenesil-1득도상응적간우재체；지질체전염법장중조체급상응적공질립분별전염지HUVEC세포，경 G418사선항성세포극륭， qRT-PCR 급 Western blot 감정 RACK1 mRNA 화단백적표체。결과RACK1진핵표체재체화RNA간우재체구건성공。중조체경지질체법전염HUVEC 48 h후，경G418사선3주득도세포항성극륭，qRT-PCR급Western blot결과증실료과표체재체화간우재체능유효적증강화침묵HUVEC세포중RACK1적표체。결론성공건립료RACK1과표체화저표체HUVEC세포계。
Objective To establish several human umbilical vein endothelial cell ( HUVEC ) strains with over-ex-pression 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 cDNA sequence of RACK1 gene was amplified and inserted into pIRES2-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 plas-mid pGenesil-1 .The HUVEC cells were transfected with these plasmids and screened by using G 418 .And the expression 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 RACK 1 were constructed success-fully.After a 48 h transfection of HUVEC cells with the recombinant vectors and G 418 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 RACK 1 have been successfully established .