PD-1敲除及GPC3修饰的嵌合抗原受体T细胞治疗肝癌的实验研究

目的 构建程序死亡受体1(PD-1)敲除及磷脂酰肌醇蛋白多糖-3(GPC3)修饰的嵌合抗原受体T细胞(GPC3-PD1gRNA-CART cells),研究其对肝癌细胞株HepG2的体外杀伤作用,以及其对肝癌动物模型的体内抗肿瘤作用.方法 制备GPC3-PD1gRNA-CART细胞,用流式细胞仪检测PD-1、GPC3 ...

Effect of PD-1 knockout and GPC3 modified chimeric antigen receptor T cells on liver cancer

Objective To study the cytotoxicity of programmed cell death protein 1(PD-1)knockout and glypican-3(GPC3)modified chimeric antigen receptor T cells(GPC3-PD1gRNA-CART cells)on the human hepatoma cell line HepG2 in vitro and the anti-tumor effect of GPC3-PD1gRNA-CART cells on the severe immunodeficiency mouse model of liver cancer in vivo.Methods GPC3-PD1gRNA-CART cells were prepared and the expression of GPC3 CARs and PD-1 were analyzed by flow cytometry.In in vitro experiment,the cytotoxicity of GPC3-PD1gRNA-CART cells stimulated with concanavalin A(ConA)on HepG2 cells was observed by LDH assay,and interferon-γ(IFN-γ)level was tested by ELISA when co-culturing the GPC3-PD1gRNA-CART cells with HepG2 cells for 18 h at different effect/target ratios(4:1,2:1,1:1).GPC3 CART and ConA-stimulated GPC3 CART were set up as control.The severe immunodeficiency mice(NDG)subcutaneous tumor model was established to assess anti-tumor effect of the GPC3-PD1gRNA-CART cells in vivo.In the subcutaneous tumor model study,there were three groups:the model group and two experiment groups(GPC3-PD1gRNA-CART group and GPC3 CART group),which were received the subcutaneous injection of HepG2 cells(1×10^6).When the diameter of tumor tissue reached 3 mm,GPC3-PD1gRNA-CART,GPC3 CART and model groups were intravenously injected with 5×10^6 GPC3-PD1gRNA-CART cells,5×10^6 GPC3 CART and 0.9% NaCl in 0.2 ml respectively,once a week for 3 weeks.The tumor volume was calculated at a 2-day interval and the pathological changes in the tumor tissues were observed for comparison.Results The GPC3 CAR expression rate of the GPC3-PD1gRNA-CART cells was more than 98.8%.The expression rate of PD-1 in GPC3 CART cells was 11.1%,which was increased to 92.1%after the stimulation with ConA,while the expression rate of PD-1 in GPC3-PD1gRNA-CART cells was only 60%,confirming the effective knockout of PD-1 gene.In in vitro experiments,the killing effect and IFN-γlevel of GPC3-PD1gRNA-CART cells stimulated by ConA on HepG2 cells were gradually increased at the effect/target ratios of 1:1,2:1 and 4:1.At the same effect/target ratio,the killing effect and the IFN-γlevel of the GPC3-PD1gRNA-CART cells stimulated with ConA on HepG2 cells were significantly higher than that of the GPC3 CART cells stimulated by Con A(P<0.05),indicating that PD-1 gene knockout in CART could reverse the inhibitory function of CART cells.The results in in vivo experiments showed that the tumor volumes in GPC3-PD1gRNA-CART group shrank 87.30%,and there was a significant difference when comparing with GPC3 CART group(69.03%)and the model group(P<0.05).The pathological changes of tumor tissues showed that the tumor area in the experiment groups was significantly decreased,and the effect of GPC3-PD1gRNA-CART group was better than GPC3 CART group.Conclusion GPC3-PD1gRNA-CART cells can effectively kill liver cancer cells in vitro and in vivo,providing a potential strategy for liver cancer therapy.