靶向EpCAM的CAR-T细胞抗肝癌细胞的体外杀伤性研究  

EpCAM-targeting CAR-T cells against hepatoma cells in vitro

作　　者：王松 张炳兰 张秉强[1] WANG Song;ZHANG Binglan;ZHANG Bingqiang(Department of Gastroenterology,the First Affiliated Hospital of Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学附属第一医院消化内科,重庆400016

出　　处：《陆军军医大学学报》2023年第18期1947-1954,共8页Journal of Army Medical University

基　　金：国家自然科学基金青年科学基金(81703057)。

摘　　要：目的研究靶向上皮细胞黏附分子(epithelial cell adhesion molecule,EpCAM)的嵌合抗原受体T细胞(chimeric antigen receptor-T cells,CAR-T)在体外对数种肝癌细胞的杀伤效能。方法采用流式细胞术检测4种人肝癌细胞(SK-hep1、hep-G2、HuH7和BEL-7402)EpCAM的表达情况;通过慢病毒载体构建靶向EpCAM的CAR-T细胞;流式细胞术检测CAR分子的表达情况并分析CAR-T的CD3/4/8表型;为分析杀伤效应,将效应细胞和靶细胞共培养后,收集上清液,通过乳酸脱氢酶(lactate dehydrogenase,LDH)释放实验检测LDH释放情况,酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测细胞因子IFN-γ和TNF-α的释放情况。结果EpCAM在hep-G2、HuH7和BEL-7402中高表达,而在SK-hep1中不表达。流式细胞术表明靶向EpCAM-CAR在慢病毒感染后的T细胞中表达率高达43.21%。流式细胞术分析CAR-T细胞CD3/4/8表型,高达95.1%的外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)为CD3阳性,其中CD8+T细胞占61.40%、CD4^(+)T细胞占32.13%。LDH释放实验提示CAR-T对EpCAM阳性人肝癌细胞hep-G2、HuH7和BEL-7402有更强的直接杀伤能力(P<0.01);ELISA实验提示EpCAM阳性人肝癌细胞hep-G2、HuH7和BEL-7402诱导CAR-T细胞释放更多的IFN-γ和TNF-α(P<0.05,P<0.01),而对于EpCAM阴性的SK-hep1肿瘤细胞,CAR-T组与未转染T细胞对照组IFN-γ和TNF-α的释放量无显著差异,此外,这2种细胞因子水平随着E∶T比值的增加而增加。结论相对未转染的T细胞,在体外,EpCAM-CAR-T细胞对EpCAM阳性的肝癌细胞具有更强的杀伤能力,能使其释放出更多的LDH、IFN-γ和TNF-α。Objective To investigate the killing efficacy of chimeric antigen receptor T(CAR-T)cells targeting epithelial cellular adhesion molecule(EpCAM)in several hepatoma cell lines.Methods Flow cytometry was used to detect the expression of EpCAM in hepatoma cells(SK-hep1,Hep-G2,HuH7 and BEL-7402).After EpCAM-targeting CAR-T cells were constructed using lentiviral vectors with CAR genes,flow cytometry was used to detect the expression of EpCAM-CAR in the obtained cells,and to analyze the CD3/4/8 phenotype of CAR-T cells without transfection of lentiviral vectors.Then the effector cells and target cells were co-cultured,and LDH release assay and ELISA were used to detect the release of LDH and major cytokines in the supernatant.Results EpCAM was highly expressed in Hep-G2,HuH7 and BEL-7402 cells,but not in SK-hep1 cells.Flow cytometry showed that the expression rate of EpCAM-CAR was 43.21%in CAR-T cells after lentivirus infection.Flow cytometry also indicated that up to 95.1%of peripheral blood mononuclear cells(PBMCs)were CD3 positive,of which 61.40%were CD8^(+)T cells and 32.13%were CD4+T cells.LDH release assay revealed that the obtained CAR-T cells had stronger direct killing ability on EpCAM-positive hepatoma cell lines Hep-G2,HuH7,and BEL-7402.ELISA showed that EpCAM-positive target cells Hep-G2,HuH7,and BEL-7402 induced CAR-T cells to release more IFN-γand TNF-α.For SK-hep1 cells with negative EpCAM expression,IFN-γand TNF-αreleases were not significantly different between the CAR-T group and the control group,and the levels of these 2 cytokines werw increased with the increase of E∶T ratio.Results Compared with ordinary CAR-T cells,EpCAM-targeting CAR-T cells have a stronger killing effect on EpCAM-positive hepatocellular carcinoma cells.

关 键 词：上皮细胞黏附分子 嵌合抗原受体T细胞 CAR-T 肝癌 

分 类 号：R394.3[医药卫生—医学遗传学] R73-362[医药卫生—基础医学] R735.7