目的：探讨IL-7的诱导表达对靶向磷脂酰肌醇蛋白聚糖3（GPC3）嵌合抗原受体基因修饰T淋巴细胞（CAR-T细胞） 的增殖和体外抗肿瘤活性的影响。方法：通过无缝克隆将GPC3 CAR序列片段插入GV400载体的BamHⅠ/EcoRⅠ位置，构建第 二代CAR慢病毒载体GPC3-BBZ及GPC3-BBZ-NFAT-IL-7，以293T细胞包装相应的慢病毒载体后，感染人T细胞制备CAR-T细 胞。实验分为未转导T细胞（NT）组、GPC3- BBZ CAR-T细胞组、GPC3-BBZ-NFAT-IL-7 CAR-T细胞组。采用流式细胞术检测各 组CAR-T细胞中CAR的表达水平，qPCR 法检测经 GPC3 蛋白激活的 CAR-T 细胞中 IL-7 mRNA的表达水平，细胞计数法检测 CAR-T细胞在GPC3抗原刺激下的增殖能力，ELISA检测CAR-T细胞在受到肿瘤细胞刺激后IL-7、IFN-γ和TNF-α的分泌水平。 应用实时细胞分析（RTCA）技术检测CAR-T细胞对人肝癌Huh-7细胞的杀伤作用。结果：成功构建慢病毒载体GPC3-BBZ和 GPC3-BBZ-NFAT-IL-7，制备出靶向GPC3 的 CAR-T 细胞。经 GPC3 抗原激活后，GPC3-BBZ-NFAT-IL-7 CAR-T细胞可有效表 达IL-7 mRNA（P < 0.01），其表现出更强的增殖能力（P < 0.05）。与GPC3-BBZ CAR-T 细胞相比，GPC3-BBZ-NFAT-IL-7 CAR-T 细胞与GPC3阳性靶细胞Huh-7细胞共培养后，分泌更高水平的IL-7、IFN-γ和TNF-α（P < 0.01或P < 0.001）。RTCA结果显示， GPC3-BBZ-NFAT-IL-7 CAR-T细胞对GPC3阳性Huh-7细胞的杀伤活性显著高于GPC3-BBZ CAR-T细胞（P < 0.05）。结论：成 功制备可诱导表达IL-7的靶向GPC3的CAR-T细胞，IL-7的诱导表达增强靶向GPC3 CAR-T细胞的免疫活性，在体外展现出较强的 肿瘤细胞杀伤能力。

Objective: To explore the effects of induced IL-7 expression on proliferation and in vitro antitumor activities of glypican-3 (GPC3)- specific chimeric antigen receptor gene modified-T (CAR-T) cells. Methods: The GPC3 CAR sequence fragment was inserted into the BamHⅠ/EcoRⅠs ite of the GV400 vector using seamless cloning, constructing second-generation CAR lentiviral vectors GPC3-BBZ and GPC3-BBZ-NFAT-IL-7. The lentiviruses were packaged with 293T cells and transfected into healthy human T cells to prepare CAR-T cells， which were divided into non-transduced T cell (NT) group, GPC3-BBZ CAR-T cell group and GPC3-BBZ-NFAT-IL-7 CAR-T cell group. The expression of CAR in CAR-T cells of each group was determined by flow cytometry. IL-7 mRNA expression level in CAR-T cells activated by GPC3 protein was determined by qPCR. The proliferation ability of CAR-T cells under GPC3 antigen stimulation was evaluated by cell counting. The secretion levels of IL-7, IFN-γ, and TNF-α by CAR-T cells after stimulation with tumor cells was determined using ELISA. The cytotoxicity of CAR-T cells against human hepatocellular carcinoma Huh-7 cells was tested by real-time cell analyzer (RTCA). Results: Lentiviral vectors GPC3-BBZ and GPC3-BBZ-NFAT-IL-7 were successfully constructed, and GPC3-specific CAR-T cells were prepared. After activation with GPC3 antigen, GPC3-BBZ-NFAT-IL-7 CAR-T cells effectively expressed IL-7 mRNA (P < 0.01) and exhibited stronger proliferation capacity (P < 0.05). Compared with GPC3-BBZ CAR-T cells, GPC3-BBZ-NFAT-IL-7 CAR-T cells co-cultured with GPC3-positive Huh-7 cells secreted higher levels of IL-7, IFN-γ, and TNF-α (P < 0.01 or P < 0.001). The RTCA results showed that the cytotoxicity of GPC3-BBZ-NFAT-IL-7 CAR-T cells against GPC3-positive Huh-7 cells was significantly higher than that of GPC3-BBZ CAR-T cells (P < 0.05). Conclusion: GPC3-specific CAR-T cells with inducible IL-7 expression were successfully prepared, which exhibited immune activity and tumor cell killing capacity in vitro.

福建省自然科学基金项目（No. 2022J01427）；福建省肿瘤医院院内项目（No. 2021YN14）