靶向EpCAM的CAR-T细胞抗肝癌细胞的体外杀伤性研究

王松; 张炳兰; 张秉强

doi:10.16016/j.2097-0927.202205133

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靶向EpCAM的CAR-T细胞抗肝癌细胞的体外杀伤性研究

基础医学 | 更新时间：2026-01-09

- 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
- 陆军军医大学学报 2023年45卷第18期页码：1947-1954
- 作者机构：
  
  400016 重庆，重庆医科大学附属第一医院消化内科
- 作者简介：
  
  [ 张秉强， E-mail:zhbingqiang@163.com ZHANG Bingqiang, E-mail:zhbingqiang@163.com
- 基金信息：
  
  国家自然科学基金青年科学基金(81703057)
- DOI：10.16016/j.2097-0927.202205133
  中图分类号： R394.3;R73-362;R735.7
- 收稿：2022-05-24，
  
  修回：2023-03-28，
  
  纸质出版：2023-09-30
- 稿件说明：
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王松, 张炳兰, 张秉强. 靶向EpCAM的CAR-T细胞抗肝癌细胞的体外杀伤性研究[J]. 陆军军医大学学报, 2023,45(18):1947-1954.

Song WANG, Binglan ZHANG, Bingqiang ZHANG. EpCAM-targeting CAR-T cells against hepatoma cells in vitro[J]. Journal of Army Medical University, 2023, 45(18): 1947-1954.
王松, 张炳兰, 张秉强. 靶向EpCAM的CAR-T细胞抗肝癌细胞的体外杀伤性研究[J]. 陆军军医大学学报, 2023,45(18):1947-1954. DOI： 10.16016/j.2097-0927.202205133.

Song WANG, Binglan ZHANG, Bingqiang ZHANG. EpCAM-targeting CAR-T cells against hepatoma cells in vitro[J]. Journal of Army Medical University, 2023, 45(18): 1947-1954. DOI： 10.16016/j.2097-0927.202205133.

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(

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(

0.05

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。

Abstract

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-γ and TNF-α. For SK-hep1 cells with negative EpCAM expression

IFN-γ and TNF-α releases 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

Keywords

references

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