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

摘要

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.%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。