运用慢病毒系统构建稳定表达CD19-FLUC-GFP的4T1细胞系  

Construction of 4T1 Cell Line Stably Expressing CD19-FLUC-GFP Using Lentivirus System

作　　者：郭雨洁 程奕宁 汪洋 刘滨磊 GUO Yujie;CHENG Yining;WANG Yang;LIU Binlei(School of Bioengineering and Food Science,Hubei Univ.of Tech.,Wuhan 430068,China)

机构地区：[1]湖北工业大学生物工程与食品学院,湖北武汉4300668

出　　处：《湖北工业大学学报》2025年第2期68-72,共5页Journal of Hubei University of Technology

摘　　要：为构建稳定表达CD19、萤火虫荧光素酶(FLUC)和绿色荧光蛋白(GFP)的乳腺癌4T1细胞系,以用作CD19嵌合抗原受体T细胞(CAR-T)的靶细胞模型,通过提取质粒、慢病毒包装及浓缩,感染4T1细胞后,经嘌呤霉素筛选和有限稀释法获得单克隆稳转细胞株4T1-CD19-FLUC-GFP。实时定量PCR和流式细胞术检测显示,4T1-CD19-FLUC-GFP细胞中CD19的mRNA和膜蛋白表达水平显著高于对照组4T1细胞。体内实验中,腋下植瘤4T1-CD19-FLUC-GFP细胞一周后,活体成像仪可检测到腋下部位的荧光信号。结果表明,成功构建了稳定表达CD19-FLUC-GFP的4T1细胞株,为CAR-T细胞抗肿瘤疗效机制研究提供了可靠的靶细胞模型和研究基础。This study aims to construct a breast cancer 4T1 cell line stably expressing CD19,firefly luciferase(FLUC),and green fluorescent protein(GFP)to serve as a target cell model for CD19 chimeric antigen receptor T cells(CAR-T).Through plasmid extraction,lentiviral packaging,and concentration,4T1 cells were infected and subsequently subjected to puromycin selection and limited dilution to obtain the monoclonal stable cell line 4T1-CD19-FLUC-GFP.Quantitative real-time PCR and flow cytometry analysis revealed that the mRNA and membrane protein expression levels of CD19 in 4T1-CD19-FLUC-GFP cells were significantly higher than those in the control 4T1 cells.In vivo experiments showed that one week after the implantation of 4T1-CD19-FLUC-GFP cells into the axilla,fluorescence signals in the axillary region could be detected using an in vivo imaging system.The results demonstrated the successful construction of a 4T1 cell line stably expressing CD19-FLUC-GFP,providing a reliable target cell model and research foundation for investigating the anti--tumor efficacy mechanisms of CAR-T cells.

关 键 词：4T1细胞 慢病毒 CD19 绿色荧光蛋白 萤火虫荧光素酶 

分 类 号：R737.9[医药卫生—肿瘤]