癌变·畸变·突变2018，Vol.30Issue(1)：14-19,6.DOI:10.3969/j.issn.1004-616x.2018.01.003
SREBP-1c基因过表达细胞模型的构建及对HepG2细胞脂滴含量的影响
Construction of SREBP-1c-overexpression cells and its effect on lipid accumulation in HepG2 cells
摘要
Abstract
OBJECTIVE:Construction of a recombinant lentiviral vector overexpressed SREBP-1c,its transfection into human hepatocellular carcinoma cell lines (HepG2) with high transfection efficiency and its effect on cellular lipid storage.METHODS:SREBP-1c gene was cloned from pCMV5-HA-SREBP-1c by polymerase chain reaction and identified by gene sequencing.The SREBP-1c gene was cloned to lentiviral expression vector (pLenti6.3/V5) by recombing DNA technology.The 293T cells were co-transfected with lentiviral packaged systems and the SREBP-1c plasmid by Lipofectamine2000 reageant to package the lentiviral particles,viral supernatant was collected,HepG2 cells were then infected by pLenti6.3-SREBP-1c.After puromycin screening,HepG2 cells were constructed.Real time PCR and Western blot assays were performed to analyzed SREBP-1c expression levels.Cellular lipid storage was detected under fluorescent microscopy and the triacylglyceride(TAG) content by TLC analysis in SREBP-1c-overexpressing HepG2 cells.RESULTS:pLenti6.3-SREBP-1c was successfully constructed and verified by restriction analysis and sequencing.SREBP-1c gene expression level in HepG2 cells with SREBP-1c gene transfection was 11.4 times higher than the control.SREBP-1c protein level in HepG2 cells with SREBP-1c gene transfection was 3.2 times higher than the control.The number and size of lipid droplets (LDs) was increased,and a higher concentration of TAG was found in SREBP-1coverexpressing cells compared with the control (P＜0.01).CONCLUSION:Recombinant lentiviral vector pLenti6.3-SREBP-1c and SREBP-1c-overexpressing cell strain was successfully constructed.The high protein and mRNA levels of SREBP-1c were assessed after HepG2 cells were transfected.SREBP-1c increased the lipid storage of HepG2 cells.The constructed cells can become a useful tool for lipid metabolism research of liver carcinoma.关键词
SREBP-1基因/慢病毒载体/人肝癌细胞HepG2/基因转染/脂滴Key words
SREBP-1c gene/lentiviral vector/HepG2 cells/gene transfection/lipid droplets分类
生物科学引用本文复制引用
刘芳,秦双红,赵燕霞..SREBP-1c基因过表达细胞模型的构建及对HepG2细胞脂滴含量的影响[J].癌变·畸变·突变,2018,30(1):14-19,6.基金项目
国家自然科学基金项目(81700766) （81700766）
