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

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

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 CAR的表达情况。体外实验中,设立不同的效靶比(1:1、2:1、4:1),用LDH法测定刀豆蛋白刺激后的GPC3-PD1gRNA-CART细胞体外对HepG2细胞株的杀伤率,检测刀豆蛋白刺激后的GPC3-PD1gRNA-CART细胞和HepG2细胞株共孵育18 h后的细胞培养上清液中IFN-γ的释放水平。设置受刀豆蛋白A刺激的和未受刀豆蛋白A刺激的GPC3修饰的CART细胞(GPC3 CART cells)作为对照组。于重度免疫缺陷小鼠(NDG小鼠)皮下注射HepG2细胞建立肝癌动物模型,观察GPC3-PD1gRNA-CART细胞体内抑瘤作用。实验分为模型组、实验组(GPC3-PD1gRNA-CART组、GPC3 CART组),模型组和实验组通过皮下注射1×10^6个HepG2细胞造模,在肿瘤长径达3 mm后实验组经尾静脉注射5×10^6个GPC3-PD1gRNA-CART细胞或GPC3 CART细胞,模型组经尾静脉注射0.9%NaCl,每周注射1次,共3周,注射体积均为0.2 ml。每隔2天观测肿瘤体积情况,治疗3周后取皮下肿瘤组织,HE染色检测肿瘤组织情况。结果流式细胞术分析结果显示,GPC3 CAR表达率为98.8%。GPC3 CART细胞PD-1的表达率为11.1%,经刀豆蛋白A刺激后,PD-1的表达率上升至92.1%,而GPC3-PD1gRNA-CART细胞的PD-1表达率仅为60%,证实了PD-1有效敲除。当效靶比为1:1、2:1、4:1时,随着效靶比的增加,刀豆蛋白刺激后的GPC3-PD1gRNA-CART细胞对HepG2的杀伤效果和IFN-γ的释放水平也随着效靶比的增加而增加。相同效靶比的情况下,刀豆蛋白A刺激后的GPC3-PD1gRNA-CART细胞对HepG2细胞的杀伤效果显著高于刀豆蛋白A刺激后的GPC3 CART细胞对HepG2细胞的杀伤效果(P<0.05),且刀豆蛋白A刺激后的GPC3-PD1gRNA-CART细胞和HepG2细胞共孵育后IFN-γ的释放水平也显著高于刀豆蛋白A刺激后的GPC3 CART细胞和HepG2细胞共孵育后IFN-γ的释放水平(P<0.05),证明PD-1的敲除可以逆转CART细胞的功能抑制。治疗3周后GPC3-PD1gRNA-CART组抑瘤率为87.30%,与GPC3 CART组(69.03%)和模型组相比均有显著性差异(P<0.05)。肿瘤组织HE染色检测结果显示,相比模型组,实验组肿瘤细胞显著减少,GPC3-PD1gRNA-CART组治疗效果优于GPC3 CART组。结论GPC3-PD1gRNA-CART细胞可解决肿瘤逃逸问题,能于体内外高效靶向杀伤肿瘤细胞,为后续肝癌治疗提供技术基础。

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.