靶向CD19嵌合抗原受体T细胞体外构建、扩增及抗肿瘤活性研究

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anti-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anti-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3.2×10⁸ 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，anti-CD19-CAR-T细胞阳性率达90.57%。经检测，anti-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anti-CD19-CAR慢病毒包装系统，成功构建靶向CD19抗原的CAR-T细胞，能特异性杀伤CD19+肿瘤细胞。

In vivo onstruction,expansion and anti-tumor activity of anti-CD19 chimeric antigen receptor T cells #br#

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 3.2×10⁸ 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.