过表达葡萄糖调节蛋白78人乳腺癌细胞系HS578T、MDA-MB-231的构建

Construction of human breast cancer cell lines HS578T and MDA-MB-231 overexpressing GRP78

目的构建过表达内质网应激标志蛋白葡萄糖调节蛋白78(Glucose-regulated protein 78,GRP78)的人乳腺癌细胞系HS578T、MDA-MB-231,为探讨GRP78在乳腺癌发生发展中的作用机制提供基础。方法采用分子克隆技术构建表达GRP78的慢病毒载体pCDH-CMV-GRP78-HA-EF1-Puro质粒,采用PCR法扩增质粒,后使用EcoRⅠ、NotⅠ限制性内切酶切割质粒,对酶切产物进行测序鉴定,成功构建pCDH-CMV-GRP78-HA-EF1-Puro质粒。取对数生长期293FT细胞系(克隆分离株),在培养皿中加入pCDH-CMV-GRP78-HA-EF1-Puro混合液,培养48 h收集含GRP78慢病毒颗粒的上清液,保存备用。另取对数生长期人乳腺癌细胞系HS578T及MDA-MB-231,置于含GRP78慢病毒颗粒上清液的培养基中培养,培养48 h时在培养基中加入1∶500稀释嘌呤霉素,筛选GRP78过表达的HS578T、MDA-MB-231细胞系。加入嘌呤霉素培养48 h采用Western Blotting法检测HS578T、MDA-MB-231细胞GRP78。采用激光共聚焦显微镜对HS578T、MDA-MB-231细胞中GRP78蛋白进行定位。结果构建表达GRP78的慢病毒载体pCDH-CMV-GRP78-HA-EF1-Puro质粒。GRP78过表达的HS578T、MDA-MB-231细胞系在78 kD左右可见明显GPR78蛋白电泳条带。HS578T细胞系中存在GRP78蛋白表达,主要分布在细胞质中。结论成功构建GRP78过表达的MDA-MB-231、HS578T细胞系,GRP78主要表达于细胞质中。

Objectives To construct human breast cancer cell lines HS578T and MDA-MB-231 that overexpress the endoplasmic reticulum stress marker glucose-regulated protein 78(GRP78),and to provide a basis for exploring the mech‐anism of GRP78 in the development and progression of breast cancer.Methods The lentiviral vector pCDH-CMV-GRP78-HA-EF1-Puro plasmid expressing GRP78 was constructed by molecular cloning technology,and the plasmid was amplified by PCR method.Then the plasmid was cut by EcoRⅠand NotⅠrestriction enzymes,and the digestion products were identified by sequencing.The pCDH-CMV-GRP78-HA-EF1-Puro plasmid was constructed successfully.293FT cell line(clonal isolate)in the logarithmic growth phase was added into a culture dish with pCDH-CMV-GRP78-HA-EF1-Puro mixture,and was cultured for 48 h;the supernate containing GRP78 lentiviral particles was collected and stored for later use.In addition,human breast cancer cell lines HS578T and MDA-MB-231 in the logarithmic growth phase were cultured in the medium containing supernate of GRP78 lentiviral particles.After 48 h culture,1:500 diluted purinycin was added to the medium,and the cell lines of HS578T and MDA-MB-231 with overexpression of GRP78 were screened.The GRP78 of HS578T and MDA-MB-231 cells was detected by Western blotting after being cultured with purinamycin for 48 h.The GRP78 protein in HS578T and MDA-MB-231 cells was localized by confocal laser microscopy.Results The cell lines of HS578T and MDA-MB-231 with overexpression of GRP78 showed obvious GPR78 protein electrophoretic bands around 78 kd.There was GRP78 protein expression in HS578T cells,which was mainly distributed in the cytoplasm.Conclusions We successfully constructed MDA-MB-231 and HS578T cell lines with GRP78 overexpression,and GRP78 was mainly ex‐pressed in the cytoplasm.