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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。
Objective To establish an active RACK1 overexpression and low expression human umbilical vein endothelial cell line (HUVEC), and to provide an effective molecular mechanism for RACK1 in arrhythmia. Methods The full-length cDNA sequence of RACK1 gene was amplified and inserted into pIRES2-EGFP to obtain the over-expression vector pIRES2-EGFP-RACK1. At the same time, three complementary DNA sequences and one pair of negative control sequences were designed and synthesized. Subcloned into p Genesil-1 to obtain the corresponding interference vector; recombinant plasmid and corresponding empty plasmid were transfected into HUVEC cells by lipofection method, and identified by G418 resistant cell clone, qRT-PCR and Western blot to identify RACK1 mRNA and protein expression. Results RACK1 eukaryotic expression vector and RNA interference vector was successfully constructed. HUVECs were transfected into HUVECs by lipofectamine for 48 h, then the cell clones were screened by G418 for 3 weeks. The results of qRT-PCR and Western blot showed that the over-expression vector and the interference vector can effectively enhance and silence RACK1 expression. Conclusion The RACK1 overexpression and low expression HUVEC cell lines were successfully established.