嵌合抗原受体修饰的CD19-CAR-T的体外构建、扩增及初步功能鉴定

In vitro construction and amplification and primary functional analysis of anti-CD19 chimeric antigen receptor(CD19-CAR) modified T cells

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p CDH-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 constructed into p CDH-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 cytotoxity against CD19＋Ramos cells. And this report might provide an experimental evidence for clinical treatment of CD19＋B cell neoplasmas.