目的：探讨shRNA靶向抑制CD38的方法能否增强抗CD38 CAR-T细胞的抗癌功能。方法：构建shRNA靶向抑制CD38 的抗 CD38 CAR-T 细胞的 CAR 分子，利用逆转录病毒载体包装成功后转导人原代 T 细胞，制备 CAR-T 细胞。实验分为shRNA1 CD38 CAR-T 组、shRNA2 CD38 CAR-T 组和对照组（shR-NC-CD38 CAR-T 细 胞）。 采 用 qPCR 法 检 测 CAR-T 细胞CD38 mRNA相对表达水平，计算CAR-T细胞培养0～14 d的增殖倍数，CFSE法检测CAR-T细胞与人Burkitt淋巴瘤细胞Raji-luc或人多发性骨髓瘤外周血B 淋巴细胞 RPMI-8226-luc 共培养时的增殖情况，荧光素酶化学发光法检测CAR-T细胞在不同效靶比（1∶1、1∶2、1∶4、1∶8）时对Raji-luc和RPMI-8226-luc细胞的杀伤效率，ELISA法检测CAR-T细胞杀伤Raji-luc或RPMI-8226-luc细胞时上清液中IFN-γ水平，FCM检测CAR-T细胞表面耗竭T细胞生物标志物PD-1的表达水平。结果：shR-NC-CD38 CAR、shRNA1-CD38 CAR 和 shRNA2-CD38 CAR 逆转录病毒载体的滴度均为 1′107拷贝/mL，转导 T 细胞后，shR-NC-CD38 CAR-T、shRNA1-CD38 CAR-T和shRNA2-CD38 CAR-T细胞的转导效率（CAR的阳性率）分别为60.3%、67.0%和57.4%。与对照组比较，shRNA2-CD38 CAR-T 组细胞中 CD38 mRNA 的表达水平显著降低（P<0.01），显示 shRNA-CD38 CAR-T 细胞构建成功。shRNA2-CD38 CAR-T 组细胞在体外培养增殖能力更强（P<0.05），对 2 种 CD38 阳性的肿瘤细胞的杀伤效率更高（均 P<0.05）、IFN-γ释放水平更高（均P<0.05）、细胞表面PD-1的表达水平更低（P<0.05）。结论：成功构建一种shRNA靶向抑制CD38的抗CD38 CAR-T细胞，其抗癌功能表现出明显的优势。

Objective:To investigate whether targeted inhibition of CD38 by shRNA can enhance the anticancer function of anti-CD38 CAR T cells. Methods: The anti-CD38 CAR molecules with targeted inhibition of CD38 by shRNA were constructed. After being successfully packaged with retroviral vector, the CAR molecules were transferred into human primary T cells to prepare CAR-T cells,which were then divided into shRNA1-CD38 CAR-T group, shRNA2-CD38 CAR-T group and control group (shR-NC-CD38 CAR-T cells). The relative expression of CD38 mRNA in CAR-T cells was detected by qPCR method, and the proliferation index of CAR-T cells cultured ex vivo for 0-14 days was calculated. The proliferation of CAR-T cells co-cultured with human Burkitt lymphoma Raji-luc cells or human multiple myeloma peripheral blood B lymphocytes RPMI-8226-luc was detected by CFSE method. The killing efficiency of CAR-T cells against Raji-luc and RPMI-8226-luc cells at different effector-target ratios (1∶1, 1∶2, 1∶4, 1∶8) was detected by luciferase chemiluminescence. The level of IFN- γ in the supernatant of CAR-T cells co-cultured with Raji-luc cells or RPMI-8226-luc cells was detected by ELISA. The expression of PD-1, one of the T cell exhaustion biomarkers, on the surface of CAR-T cells was detected by flow cytometry. Results: The titers of shR-NC-CD38 CAR, shRNA1-CD38 CAR and shRNA2-CD38 CAR retroviral vectors were all about 1×107 copies/mL. The transduction efficiency (CAR positive rate) in cells of shR-NC-CD38 CAR-T group, shRNA1-CD38 CAR-T group and shRNA2-CD38 CAR-T group was 60.3%, 67.0% and 57.4%, respectively. Compared with the control group, the expression level of CD38 mRNA in shRNA2-CD38 CAR-T group was significantly lower (P<0.01),indicating the successful construction of shRNA-CD38 CAR-T cells. For the cells in shRNA2-CD38 CAR-T group, their ex vivo proliferation ability was stronger, the killing efficiency against two CD38 positive tumor cells was higher, the release level of IFN-γ was higher, and the surface expression level of PD-1 was lower, as compared with the other two groups (P<0.05). Conclusion: A novel anti[1]CD38 CAR-T cells with targeted inhibition of CD38 by shRNA was successfully constructed, which exhibited obvious advantages in antitumor function.

北京市双一流高层次科研团队科研资助项目（No.1000041510100）