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

癌变·畸变·突变 ›› 2018, Vol. 30 ›› Issue (1): 14-19.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

刘芳^1,2, 秦双红³, 赵燕霞³

1. 第四军医大学西京医院皮肤科, 陕西西安 710032;
2. 第四军医大学西京医院病理科, 陕西西安 710032;
3. 解放军第518医院, 陕西西安 710043

收稿日期:2017-09-25 修回日期:2017-12-13 出版日期:2018-01-30 发布日期:2018-01-30
作者简介:刘芳,E-mail:553417048@qq.com
基金资助:
国家自然科学基金项目（81700766）

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

LIU Fang^1,2, QIN Shuanghong³, ZHAO Yanxia³

1. Department of Dermatology, Xijing Hospital, the Fourth Military Medical University, Xi'an 710032;
2. Department of Pathology, Xijing Hospital, the Fourth Military Medical University, Xi'an 710032;
3. PLA 518 Hospital, Xi'an 710043, Shaanxi, China

Received:2017-09-25 Revised:2017-12-13 Online:2018-01-30 Published:2018-01-30

摘要/Abstract

摘要： 目的：构建SREBP-1c过表达HepG2细胞模型并观察其对脂滴含量的影响，为进一步深入研究肝癌脂滴代谢异常机制提供细胞模型。方法：采用DNA重组技术，将SREBP-1c基因克隆至慢病毒表达载体pLenti 6.3/V5中，并用脂质体介导法将慢病毒包装系统和带目的基因的质粒pLenti6.3-SREBP-1c共转染293T细胞，收集上清，感染HepG2细胞。通过Real-time PCR和Western blot对细胞株进行鉴定。采用免疫荧光检测HepG2细胞中脂滴的数量、大小及细胞内甘油三酯的含量。结果：成功构建了过表达SREBP-1c基因的慢病毒载体pLenti6.3-SREBP-1c，转染后HepG2细胞中可见明显的SREBP-1c蛋白表达。Real-time PCR检测结果显示转染SREBP-1c基因的HepG2细胞中SREBP-1c mRNA水平较对照组升高了约11.4倍。Western blot结果显示SREBP-1c蛋白水平较对照组提高3.2倍。免疫荧光显示过表达SREBP-1c能够明显增加HepG2细胞内脂滴的数量和大小。细胞内甘油三酯定量结果显示甘油三酯含量较对照组明显升高（P < 0.01）。结论：成功构建了SREBP-1c基因过表达的重组慢病毒载体及其稳定转染细胞株。高表达SREBP-1c的HepG2细胞增加了细胞中脂滴的数量、大小和甘油三酯的含量，可用于从脂滴代谢方面对肝癌发病分子机制的研究。

关键词: SREBP-1c基因, 慢病毒载体, 人肝癌细胞HepG2, 基因转染, 脂滴

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

中图分类号:

Q291

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.

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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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

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