稳定表达CD19的非小细胞肺癌细胞系的构建  

Construction of A549 Cell Line Stably Expressing CD19 Using PiggyBac System

作　　者：朱帅旗 张帆 詹思建 段海潇 王迪 胡翰 汪洋 刘滨磊 ZHU Shuaiqi;ZHANG Fan;ZHAN Sijian;DUAN Haixiao;WANG Di;HU Han;WANG Yang;LIU Binlei(School of Biological Engineering 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430068

出　　处：《湖北工业大学学报》2023年第2期52-56,共5页Journal of Hubei University of Technology

摘　　要：细胞系表达的CD19可被靶向CD19的嵌合抗原受体(CAR)特异性识别。为探究靶向CD19 CAR-T抗肿瘤疗效及机制提供可靠稳定的靶细胞系,运用Lipofectamine 3000 TM脂质体转染法将pPBDP-CD19和pSPBT质粒转入A549细胞。嘌呤霉素(Puro)筛选,稳定细胞株A549-CD19单克隆细胞由有限稀释法得到;RT-qPCR鉴定A549-CD19细胞系中CD19 mRNA的表达;流式细胞术检测A549-CD19中CD19的表达;MTS法比较改造前后细胞系的生长活性变化。经Puro抗性筛选和挑取单克隆,得到4个A549-CD19单克隆细胞;RT-qPCR鉴定A549-CD19细胞系均有CD19 mRNA高表达;流式检测单克隆细胞系阳性率均达到99%;MTS法检测发现A549-CD19细胞系与亲本细胞A549细胞具有一致的生长活性。运用PiggyBac转座系统构建稳定表达CD19的A549-CD19细胞系成功,为评估靶向CD19实体瘤CAR-T疗效提供靶细胞,奠定CAR-T细胞抗实体瘤疗效机制研究基础。Objective:Non-small cell lung cancer(NSCLC)A549-CD19 cell line stably expressing CD19 was constructed by PiggyBac transposon system.The CD19 expressed by the cell line can be specifically recognized and bound by the chimeric antigen receptor(CAR)targeting CD19,providing a reliable and stable target cell line for exploring the anti-tumor efficacy and mechanism of the targeting CD19 CAR-T.Methods:pPBDP-CD19 and pSPBT plasmids were transferred into A549 cells by Lipofectamine 3000TM liposome transfection,stable cell lines were screened by puromycin(Puro),A549 CD19 monoclonal cells were obtained by limited dilution method,the expression of CD19 mRNA in A549 CD19 cell line was identified by RT qPCR,the expression of CD19 in A549 CD19 was detected by flow cytometry,and the growth activity of cell line before and after modification was compared by MTS method.Results:After Puro resistance screening and selection of clones,four A549 CD19 monoclonal cells were obtained;all A549 CD19 cell lines were identified by RT qPCR to have high expression of CD19 mRNA;flow detection showed that the positive rate of monoclonal cell lines reached 99%MTS method.It was found that the growth and viability of A549 CD19 cell line was consistent with that of parent cell A549.Conclusion:The A549 CD19 cell line stably expressing CD19 is successfully constructed,which provides target cells for evaluating the efficacy of CAR T targeting CD19 solid tumor,and lays the foundation for the study of the mechanism of CD19 CAR T cell anti-solid tumor effect.

关 键 词：A549细胞 PIGGYBAC CD19 绿色荧光蛋白 

分 类 号：R153[医药卫生—营养与食品卫生学]