[摘要] 目的：根据抗CD33-scFv 序列构建CD33-CAR 修饰的NK92 细胞，探讨其对CD33+ 急性髓系白血病（acute myeloidleukemia，AML）细胞的杀伤作用。方法：通过基因合成以及分子克隆技术获得抗CD33-CAR片段，然后将其构建到慢病毒载体上，进行慢病毒包装，将得到的慢病毒转染NK92 细胞，用流式细胞术检测细胞转染效率并通过嘌呤霉素筛选得到稳定表达抗CD33-CAR的细胞系CD33-CAR-NK92，利用钙黄绿素释放法检测该细胞的杀伤作用，ELISA法检测细胞因子IFN-γ 分泌的变化。结果：成功构建pCDH-CD33-CAR 重组慢病毒质粒，慢病毒转导后约18.7%的NK92 细胞表达CD33-CAR（命名为CD33-CARNK92细胞），嘌呤霉素筛选后表达CD33-CAR的NK92 细胞比例约86.3%。CD33-CAR-NK92 细胞对CD33+AML细胞MOLM-13的杀伤作用明显高于未被基因修饰的NK92 细胞（P<0.01），而两者对CD33-肿瘤细胞JURKAT 的杀伤作用没有明显差异（P>0.05）。效靶比为2∶1 共培养6 h 后，经CD33-CAR修饰的NK92 细胞相比未被基因修饰的NK92 细胞IFN-γ 的分泌水平明显升高[ （190.97±11.52）vs（88.41±2.75）pg/ml, P<0.01]。结论：CD33-CAR-NK92 细胞能特异性识别CD33 抗原并杀伤CD33+AML细胞，其杀伤作用显著高于未被基因修饰的NK92 细胞，为进一步开展靶向CD33 的CAR-NK92 细胞治疗AML的临床转化奠定了实验基础。

[Abstract] Objective: To construct CD33-CAR modified NK92 cells based on CD33-scFv sequence, and to explore its killing effect on CD33+ AML (acute myeloid leukemia) cells. Methods: DNA fragment encoding CD33-CAR was synthesized by gene synthesis and molecular cloning technology and then cloned into lentiviral vector. Lentivirus were packaged and used to transfect NK92 cells. The transfection efficiency was detected by flow cytometry, and puromycin was used to screen NK92 cells stably expressing CD33-CAR(CD33-CAR-NK92). Killing effect of CD33-CAR-NK92 cells on AML cells in vitro was examined with calcein-AM release assays.IFN-γ secretions of NK92 cells and CD33-CAR-NK92 cells were measured by ELISA. Results: The pCDH-CD33-CAR lentiviral vector was successfully constructed. After lentiviral transfection, about 18.7% of NK92 cells express CD33-CAR (referred as CD33-CARNK92 cells). The percentage of CD33-CAR+ NK92 cells was about 86.3% after puromycin selection. In contrast to unmodified NK92 cells, significantly higher cytotoxic effect against CD33+ MOLM-13 cells was found in CD33-CAR-NK92 cells (P<0.01); however,there was no significant difference in cytotoxicity against CD33- JURKAT cells between NK92 cells and CD33-CAR-NK92 cells (P>0.05). After co-culture at an effect-target ratio of 2∶1 for 6 hours, the level of IFN-γ secreted by the CD33-CAR modified NK92 cells was significantly higher than that of the unmodified [190.97±11.52] vs [88.41±2.75]pg/ml, P<0.01). Conclusion: The CD33-CARNK92 cells could specifically recognize CD33 antigen and kill CD33+ AML cells in comparison with the unmodified NK92 cells, which provides experimental basis for clinical transformation of CD33-CAR-NK92 cells in treating AML.

国家自然科学青年基金资助项目（No.31401170）