摘要
目的:探索特异性靶向CD19的嵌合抗原受体(chimeric antigen receptor,CAR)T细胞的构建与制备方法,并研究其体外杀伤靶细胞的效果。方法:通过基因合成和分子克隆手段构建靶向CD19-CAR片段并将其插入pLenti6.3慢病毒载体,利用293FT悬浮细胞系包装慢病毒并转染人外周血单个核细胞来源的CD3^+T细胞。通过流式细胞术鉴定转染效率,并通过实时无标记细胞分析法和细胞计数试剂盒8检测靶向CD19-CAR-T细胞体外杀伤效果。结果:获得了表达抗CD19的单链抗体基因的慢病毒,病毒滴度可达3.2×10^8噬斑形成单位/ml。经慢病毒转染的CD3^+T细胞在体外培养14 d后,细胞扩增效率达(60.2±11.5)倍,靶向CD19-CAR-T细胞阳性率达90.57%。经检测,靶向CD19-CAR-T细胞均可有效杀伤CD19^+靶细胞。结论:建立了基于无血清悬浮细胞系的靶向CD19-CAR慢病毒包装系统,成功构建靶向CD19的CAR-T细胞,该细胞能特异性杀伤CD19^+肿瘤细胞。
Objective To explore the construction and preparation methods of chimeric antigen receptor(CAR)modified T cells that specifically target CD19,and to study their effects of killing target cells in vitro.Methods The CD19-CAR was constructed by gene synthesis and molecular cloning,then inserted into plenti6.3 lentiviral vector.Lentivirus was packaged using 293FT suspension cell line and transfected into CD3^+T cells derived from human peripheral blood mononuclear cells.Transfection efficiency was identified by flow cytometry,and anti-tumor activity of CD19-CAR-T cells in vitro was detected by real-time cell analysis system and cell counting kit-8.Results Lentivirus expressing the anti-CD19 single-chain variable fragment gene was obtained.The virus titer reached 3.2×10^8 plaque-forming units/ml.CD3^+T cells transfected with lentivirus had expanded(60.2±11.5)times,and the positive rate of anti-CD19-CAR T cells was as high as 90.57%after cultured for 14 d in vitro.CD19-CAR-T cell effectively killed CD19^+target cells.Conclusions CD19-CAR lentiviral packaging system based on a serum-free suspension cell line was established,and a CAR-T cell which target CD19 antigen and can specifically kill CD19-positive tumor cells was successfully constructed.
作者
郑眉
丁亚红
熊斐斐
刘雪颖
段鹏
姜凤婷
罗剑
Zheng Mei;Ding Yahong;Xiong Feifei;Liu Xueying;Duan Peng;Jiang Fengting;Luo Jian(No.4 Research Laboratory,Shanghai Institute of Biological Products Co.,Ltd,Shanghai 200051,China)
出处
《国际生物制品学杂志》
CAS
2020年第5期224-228,共5页
International Journal of Biologicals
