肝刺激因子稳定转染人肝癌细胞系的方法建立与表达鉴定

doi:10.3969/j.issn.1006-7795.2015.06.016

首都医科大学学报 ›› 2015, Vol. 36 ›› Issue (6): 924-928.doi: 10.3969/j.issn.1006-7795.2015.06.016

肝刺激因子稳定转染人肝癌细胞系的方法建立与表达鉴定

吴媛, 张静, 董凌月, 安威

首都医科大学基础医学院细胞生物学系肝脏保护与再生调节北京市重点实验室, 北京 100069

收稿日期:2015-06-11 出版日期:2015-12-21 发布日期:2015-12-18
通讯作者: 安威 E-mail:anwei@ccmu.edu.cn
基金资助:
国家自然科学基金(31371169)。

Construction and expressional identification of human hepatic cell stably transfected with HSS

Wu Yuan, Zhang Jing, Dong Lingyue, An Wei

Department of Cell Biology, School of Basic Medical Sciences, Capital Medical University, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Beijing 100069, China

Received:2015-06-11 Online:2015-12-21 Published:2015-12-18
Supported by:
This study was supported by National Natural Science Foundation of China(31371169).

摘要/Abstract

摘要： 目的建立稳定表达肝刺激因子(hepatic stimulator substance,HSS)的BEL-7402肝癌细胞系,并对其生物学特性进行鉴定。方法采用脂质体法将鉴定后的Flag-HSS-pcDNA3.0质粒转染到肝癌细胞系BEL-7402中,进行G418筛选,获得稳定转染的人肝癌细胞系。通过real-time PCR、 Western blotting和免疫荧光染色等方法鉴定HSS mRNA和蛋白质表达及亚细胞定位。结果免疫荧光染色实验证实HSS基因能在稳定转染的肝癌细胞系中表达,并定位于线粒体中。同时发现在稳定转染细胞中HSS mRNA和蛋白质的表达也均高于野生型BEL-7402细胞。结论目前已成功构建稳定转染HSS的BEL-7402肝癌细胞系。为深入研究HSS的生理功能提供实验工具和研究基础。

关键词: 肝刺激因子, 人肝癌细胞, 转染

Abstract: Objective To construct a hepatic cell stably transfected with HSS gene, and to identify its biological characteristics. Methods Flag-HSS-pcDNA3.0 was transfected into BEL-7402 cell line via FuGENE HD and G418 selection. The stably transfected cell line was identified by immunofluorescence, real-time PCR, and Western blotting. Results Immunofluorescence demonstrate the expression of HSS in the stably transfected cell line and the mitochondrial location of HSS. The expression of mRNA and protein levels were significantly increased in the hepatic cell line stably transfected with HSS gene compared with the wild type cell line. Conclusion A new hepatic cell lines transfected with HSS is successfully established, and it will provide the experiment tools and research base to in-depth study of the physiological functions of HSS.

Key words: hepatic stimulator substance, hepatic cell, transfection

中图分类号:

Q503

吴媛, 张静, 董凌月, 安威. 肝刺激因子稳定转染人肝癌细胞系的方法建立与表达鉴定[J]. 首都医科大学学报, 2015, 36(6): 924-928.

Wu Yuan, Zhang Jing, Dong Lingyue, An Wei. Construction and expressional identification of human hepatic cell stably transfected with HSS[J]. Journal of Capital Medical University, 2015, 36(6): 924-928.

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肝刺激因子稳定转染人肝癌细胞系的方法建立与表达鉴定

Construction and expressional identification of human hepatic cell stably transfected with HSS

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