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

doi:10.3969/j.issn.1004-616x.2018.01.003

Abstract

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-1c-overexpressing 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.

Key words: SREBP-1c gene, lentiviral vector, HepG2 cells, gene transfection, lipid droplets

CLC Number:

Q291

LIU Fang, QIN Shuanghong, ZHAO Yanxia. Construction of SREBP-1c-overexpression cells and its effect on lipid accumulation in HepG2 cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(1): 14-19.

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Construction of SREBP-1c-overexpression cells and its effect on lipid accumulation in HepG2 cells

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