摘要
目的:构建活化转录因子2(ATF-2)基因RNA干扰的真核表达载体,观察其对HepG2肝癌细胞株增殖及凋亡的影响。方法:根据ATF-2 mRNA序列,合成两条寡聚DNA片段,退火后克隆入质粒载体PBA-siU6,DNA测序鉴定重组质粒PBA-siATF-2。脂质体介导质粒转染HepG2细胞。Westernblot法检测ATF-2蛋白表达;MTT方法检测细胞增殖率,流式细胞术(FCM)检测细胞凋亡率。结果:ATF-2基因RNA干扰载体经测序分析证实插入64 bp序列正确无误;Westernblot法显示干扰组细胞内ATF-2蛋白表达量明显降低;ATF-2基因的下调导致HepG2细胞增殖阻滞和凋亡发生。结论:成功构建了ATF-2基因RNAi真核表达载体,下调HepG2细胞ATF-2基因表达,抑制细胞增殖,诱导细胞凋亡。
AIM：To construct an eukaryotic expression vector for RNA interference targeting activating transcription factor 2（ATF-2） gene,and explore its effect on proliferation and apoptosis of HepG2 cells.METHODS： Two complementary oligonucleotides were synthesized based on ATF-2 mRNA sequence.The annealed fragment was inserted into the vector PBA-siU6.The recombinant plasmid PBA-siATF-2 was confirmed by DNA sequencing and transfected into HepG2 cells mediated by liposome.After transfection,ATF-2 protein was detected by Western blotting.The cellular growth activity and apoptosis rate were measured by MTT assay and flow cytometry,respectively.RESULTS： Recombinant plasmid expressing siRNA targeting ATF-2 gene was confirmed by DNA sequencing.Plasmid transfection down-regulated the level of ATF-2 protein in HepG2 cells,which blocked cellular growth and induced cell apoptosis.CONCLUSION： The eukaryotic expression vector for RNA interference targeting ATF-2 gene was constructed successfully,which inhibits HepG2 cell proliferation and induces cell apoptosis.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2012年第9期985-987,共3页
Chinese Journal of Cellular and Molecular Immunology
