靶向乙型肝炎表面抗原嵌合抗原受体T细胞对肝细胞癌杀伤作用的研究  

Study on killing effect of HBsAg specific CAR-T cells on hepatocellular carcinoma

作　　者：隋明昊 王歈[2] 李崇辉[1] 卢实春[1] Sui Minghao;Wang Yu;Li Chonghui;Lu Shichun(Faculty of Hepato-Pancreato-Biliary Surgery,Chinese PLA General Hospital,Beijing 100853,China;Department of General Surgery,Xuanwu Hospital of Capital Medical University,Beijing 100053,China)

机构地区：[1]解放军总医院肝胆胰外科医学部,北京100853 [2]解放军总医院肿瘤中心实验室,北京100853 [3]首都医科大学宣武医院普外科,北京100053

出　　处：《中华肝胆外科杂志》2022年第1期51-55,共5页Chinese Journal of Hepatobiliary Surgery

基　　金：国家自然科学基金(81670590)。

摘　　要：目的:分析靶向乙型肝炎表面抗原(HBsAg)的嵌合抗原受体(CAR)-T细胞对表达HBsAg的肝癌细胞的杀伤作用。方法:构建HBsAg-CAR基因,通过慢病毒载体将其转导入T细胞(健康捐献者的血液中获取),制备HBsAg-CAR-T细胞。制备CD19-CAR-T细胞作为Mock组,未转导的T细胞为Untransduced组。上述三种效应细胞分别与肝癌细胞共同培养,检测三组细胞对肝癌细胞的杀伤作用及抗肿瘤细胞因子(肿瘤坏死因子-α、干扰素-γ、白细胞介素2等)释放水平。建立免疫缺陷NPG小鼠PLC/PRF/5肝癌皮下成瘤模型,随机分组,尾静脉注射HBsAg-CAR-T细胞(实验组,n=5)或未转导的T细胞(对照组,n=5),注射后第15天测量肿瘤体积。结果:体外培养过程中,HBsAg-CAR-T细胞具有较高的增殖能力及存活率,CAR的表达稳定。与表达HBsAg的肝癌细胞共培养后,HBsAg-CAR-T组释放的抗肿瘤细胞因子明显高于Mock组及Untransduced组,差异均有统计学意义(均P<0.05);HBsAg-CAR-T组对HBsAg表达阳性的肝癌细胞的杀伤率明显高于Mock组及Untransduced组,差异均有统计学意义(均P<0.05)。实验组NPG小鼠肿瘤体积为(250.8±62.8)mm 3,低于对照组小鼠肿瘤体积(757.5±102.6)mm 3,差异有统计学意义(P<0.05)。结论:HBsAg-CAR-T细胞能够特异性识别并杀伤HBsAg阳性肝癌细胞,并释放高水平的抗肿瘤细胞因子。Objective To analyze the anti effect of chimeric antigen receptor(CAR)-T cells targeting hepatitis B surface antigen(HBsAg)on hepatocellular carcinoma cells.Methods HBsAg-CAR gene was transduced into T cells(obtained from the blood of healthy donors)through a lentiviral vector.CD19-CAR-T cells were included as mock group,and untransduced T cells were included as control group.Cells of the three groups were co-cultured with hepatocellular carcinoma cells expressing HBsAg or not to detect the anti effect and releasing level of anti-tumor cytokines(tumor necrosis factor-α,interferon-γ,interleukin-2).Subcutaneous xenograft PLC/PRF/5 tumor model using NPG mice were established and HBsAg-CAR-T cells(experimental group,n=5)or untransfected T cells(control group,n=5)were injected through tail vein.Tumor volume was measured 15 days after injection.Results HBsAg-CAR-T cells proliferation was good under in vitro culture,and the expression rate of CAR was stable.After co-cultured with hepatocellular carcinoma cells expressing HBsAg,the level of anti-tumor cytokines released by HBsAg-CAR-T cells was significantly higher than that of the other two groups of T cells,and the difference was statistically significant(all P<0.05);the anti rate of HBsAg-CAR-T cell group on HBsAg-positive hepatocellular carcinoma cells was significantly higher than that of the other two groups,and the difference was statistically significant(all P<0.05).The tumor volume of NPG mice in the experimental group was(250.8±62.8)mm3,which was lower than that of the control group(757.5±102.6)mm3,and the difference was statistically significant(P<0.05).Conclusion HBsAg-CAR-T cells can specifically recognize and kill HBsAg-positive hepatocellular carcinoma cells and release high level of anti-tumor cytokines.

关 键 词：癌 肝细胞 乙型肝炎表面抗原 T淋巴细胞 免疫治疗 

分 类 号：R735.7[医药卫生—肿瘤]