中国肿瘤生物治疗杂志2017，Vol.24Issue(7)：748-755,8.DOI:10.3872/j.issn.1007-385x.2017.07.009
靶向肝癌的GPC3-CAR-T细胞的构建与功能鉴定
Construction of GPC3-CAR-T cell targeting hepatocellular carcinoma and identification of its function
摘要
Abstract
Objective:To construct chimeric antigen receptor (CAR)-modified T cell (CAR-T) which targets GPC3 positive hepatoma cell,and to assess its killing efficacy to the GPC3 positive hepatoma cell.Methods:To enhance expression efficiency of the CAR molecule,technique for favored codon and modification of gene sequence were selected.CAR gene targeting GPC3 antigen was designed and lentiviral vector carrying GPC3-CAR gene,pCDHGPC3-CAR,was constructed,which was transfected into T cell.Western blotting,flow cytometry,real-time cellanalysis (RTCA) and ELISA assays were used respectively to detect expression of GPC3-CAR molecule in the CAR-T cell,infection efficiency of the lentivirus to T cells,killing activity and specificity of the GPC3-CAR-T cell to the GPC3 positive hepatoma cell.Results:The gene segment in the pCDH-GPC3-CAR recombinant lentivirus plasmid,molecular mass of which is the same as the GPC3-CAR molecule,was found in a result of double enzyme digestion,indicating that the pCDH-GPC3-CAR lentivirus plasmid was successfully constructed.About 54.38％ of the GPC3T cell expressing GPC3-CAR molecule were celled as the GPC3-CAR-T cell.Killing efficacy of the GPC3-CAR-T cell to the GPC3 positive Huh-7 hepatoma cell was higher than that to the GPC3 negative SK-HEP-1 cell ([78.96±4.76]％ vs [6.87±3.15]％).IFN-y secretion efficiency of the GPC3-CAR-T cell co-cultured with the GPC3 positive Huh-7 hepatoma cell in the CAR-T group was higher than that in the Mock group ([21 371.4± 1 808.3]pg/ml vs [152.8±12.5] pg/ml),which could be one of the mechanisms of high efficacy in killing hepatoma cells.Conclusion:The GPC3-CAR-T cell targeting hepatoma cells might have high efficacy ability to secrete cytokine IFN-γand specific,higher efficient ability to kill the GPC3 positive hepatoma cell,which would lay a foundation to carry further forward pre-clinical and clinical research on the GPC3-CAR-T cell.关键词
磷脂酰肌醇蛋白聚糖-3/嵌合抗原受体T细胞/免疫治疗/肝癌Key words
glypican-3(GPC3)/chimeric antigen receptor T cells (CAR-T)/immunotherapy/hepatocellular carcinoma分类
医药卫生引用本文复制引用
吴斯玮,敖翔,郭韡,邢伟,安天琛,敖罗权,胡雪停,李战,徐祥..靶向肝癌的GPC3-CAR-T细胞的构建与功能鉴定[J].中国肿瘤生物治疗杂志,2017,24(7):748-755,8.基金项目
国家自然科学基金资助项目(No.81502434),第三军医大学校管课题资助项目(No.2014YQN06).Project suported by the National Natural Science Foundation (No.81502434),and the Foundation for Projects Administrated by the Third Military Medical University (No.2014YQN06) （No.81502434）
