目的：开发基于PiggyBac（PB）转座系统的电转染CAR-T 细胞制备方法并鉴定其体外抗肿瘤功能。方法：采用健康人外周血单个核细胞（PBMC）制备T细胞，通过分子克隆技术将CD19 基因克隆到PB质粒（转座子）中后经电转染法将转座子和转座酶质粒导入激活的T细胞中，并测定其转染效率，最后运用流式细胞术及荧光素酶发光实验评估其对人Burkitt's 淋巴瘤Raji细胞的杀伤能力。结果：电转染制备的CD19 CAR-T 细胞转染效率较高（>60%），呈剂量依赖性，且CAR-T 细胞相对于Pan-T 细胞对Raji 细胞杀伤能力显著（P<0.05）。结论：开发的PB转座系统的电转染方法可行，在体外对肿瘤细胞具有显著的杀伤能力，具备临床运用于CD19 CAR-T细胞制备的潜力。

Objective: To develop an electroporation CAR-T cell preparation method based on PiggyBac (PB) transposable system and to characterize its anti-cancer function in vitro. Methods: Healthy human peripheral blood mononuclear cells (PBMCs) were used to obtain T cells. The CD19 gene was cloned into PB plasmid (transposon) by molecular cloning technique. Then, the transposon and transposase plasmid were introduced into activated T cells by electroporation, and the transfection efficiency was measured. Finally, the ability of prepared CAR-T cells to kill human Burkitt's lymphoma Raji cells was evaluated by flow cytometry and luciferase luminescence assay. Results: The transfection efficiency of CD19 CAR-T cells prepared by electroporation was high (>60%) in a dose-dependent manner, and CAR-T cells had a significantly higher cytotoxicity to Raji cells relative to the Pan-T cell group (P<0.05).Conclusion: The developed PB transposable system is feasible for electrotransfection and has the potential for clinical application inCD19 CAR-T cell preparation due to its significant in vitro cancer cell killing ability.

北京市双一流高层次人才科研启动经费资助项目（No.9011451310032）；北京中医药大学新教师启动基金资助项目（No.1000061223998）