目的： 探索一种特异性靶向 CD19 分子的嵌合抗原受体修饰的 CD19-CAR-T 的构建方法，并明确其体外杀伤 靶细胞的效果。 方法： 运用分子克隆技术，将 PCR 获得的 CD19-CAR 片段构建到 pCDH-GFP 慢病毒载体上，利用包装 的慢病毒颗粒转染供者 CD3 + T 细胞，通过流式细胞术及 PCR鉴定转染效率，并通过 7-AAD染色鉴定扩增得到的CD19- CAR-T细胞体外杀伤CD19 + Ramos靶细胞的效果。 结果： 经慢病毒转染T细胞在体外培养 10 d 后，CD3 + T 扩增达（78.8± 23.2）倍， （58.3±5.4）%的 CD3 + T 细胞表达 GFP，CD19-CAR-T 体外杀伤 CD19 + Ramos 靶细胞在效靶比5∶1时效率为（57.4± 9.3）%。 结论： 本实验成功建立了一种有效的体外构建及扩增CD19-CAR-T的方法，且具有明显靶向性，为CD19 + B细胞肿瘤临 床治疗提供实验依据。

Objective: To establish a chimeric antigen receptor（CAR）modified T cells specifically targeting CD19 molecule (CD19- CAR-T cells) and to testify their in vitro killing effect on target cells. Methods: CD19-CAR fragments yielded by PCR were construct- ed into pCDH-GFP lentiviral vectors by molecular cloning technology. The packaged lentiviral particles were transducted into CD3 + T cells of donors. Transduction efficiency was measured by flow cytometry and PCR. The in vitro cytotoxicity of obtained CD19- CAR-T cells against CD19 + Ramos cells was tested by 7-AAD staining. Results: The amplification folds of CD3 + T cells increased to (78.8± 23.2) folds after in vitro culture for 10 days, and about (58.3±5.4)% cells expressing GFP. About (57.4±9.3)% CD19 + Ramos cells were specifically killed by the CD19-CAR-T cells in vitro at the E∶T ratio of 5∶1. Conclusion: This study successfully established an effective method for constructing and amplifying CD19-CAR-T cells in vitro, which showed profound efficiency and specific cyto- toxity against CD19 + Ramos cells. And this report might provide an experimental evidence for clinical treatment of CD19 + B cell neo- plasmas.

江苏省社会发展-临床前沿技术资助项目（No. BE2016809）；南京市科技发展计划项目资助（No. 201503011）