嵌合受体T淋巴细胞诱导癌胚抗原阳性的特异性胃癌细胞凋亡

[T lymphocytes with chimeric receptor induce carcinoembryonic antigen-positive specific gastric carcinoma cells apoptosis].

作者信息

Xu Hong-yong, Xu Li, Gao Jian-hong, Li Kai-zong, Dou Ke-feng

机构信息

Hepatobiliary General Surgery, Hospital No. 451 of Chinese People's Liberation Army, Xi'an 710054, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2007 Apr 17;87(15):1053-7.

PMID:17672971

Abstract

OBJECTIVE

To develop a method to generate T lymphocytes that identify tumor specific carcinoembryonic antigen (CEA), and induce anti-tumor response such as apoptosis.

METHODS

Peripheral blood samples were collected from 10 healthy persons and peripheral blood mononuclear cells (PBMCs) were isolated. 1, then CD8(+) T cells were isolated from the PBMCs by magnetic activated cell sorting (MACS) using magnetic beads-conjugated anti-CD8 monoclonal antibodies. Then the recombinant vector anti-CEA-scFv-CD3zeta-pcDNA3.0 was transfected into the CD8(+) T cells by lipofectamine 2000 and T lymphocytes with chimeric receptor were generated and cultivated. The T lymphocytes' activation was assessed by detecting the expression of CD69, an early signal of T cell activation. Human gastric carcinoma cells of the lines HGC-27 (CEA(+)) and SGC-7901 (CEA(-)) were cocultivated with the T lymphocytes with chimeric receptor. Flow cytometry (FCM) with annexin V-FITC/PI double staining was used to detect the expressions of annexin V, a signal of cell apoptosis, so as to observe the apoptosis of the gastric carcinoma cells. T cells activated by phytohemagglutinin (PHA) were used as positive controls, and T cells transfected with blank vector pcDNA3.0 were used as negative controls.

RESULTS

T lymphocytes with chimeric receptor directed towards CEA(+) gastric carcinoma cells were generated. 12 and 24 hours after the co-culture of these T cells and HCG-27 cells, the CD69 expression rates were 40.5% +/- 3.4% and 48.3% +/- 2.8% respectively. However, the CD69 expression rates of the HGC-27 and SGC-7901 cells transfected with the T lymphocytes transfected with blank vector were both 0%. FCM showed that the apoptotic rates of the CEA(+) gastric tumor HGC-27 cells was 47.8% +/- 4.2%, significantly higher than that of the CEA(-) gastric tumor SGC-7901 cells (18.7% +/- 2.8%, P < 0.05).

CONCLUSION

T lymphocytes with chimeric receptor targeting CEA specifically identify CEA positive gastric carcinoma cells and promote the apoptosis thereof, thus providing a promising method of cellular immunotherapy for gastric carcinomas.

摘要

目的

开发一种生成可识别肿瘤特异性癌胚抗原（CEA）的T淋巴细胞并诱导诸如凋亡等抗肿瘤反应的方法。

方法

从10名健康人采集外周血样本并分离外周血单个核细胞（PBMC）。然后，使用磁珠偶联的抗CD8单克隆抗体通过磁激活细胞分选（MACS）从PBMC中分离CD8(+) T细胞。接着，用脂质体2000将重组载体抗CEA - scFv - CD3ζ - pcDNA3.0转染到CD8(+) T细胞中，生成并培养带有嵌合受体的T淋巴细胞。通过检测T细胞激活的早期信号CD69的表达来评估T淋巴细胞的激活情况。将人胃癌细胞系HGC - 27（CEA(+)）和SGC - 7901（CEA(-)）与带有嵌合受体的T淋巴细胞共培养。采用膜联蛋白V - FITC/PI双染的流式细胞术（FCM）检测细胞凋亡信号膜联蛋白V的表达，以观察胃癌细胞的凋亡情况。用植物血凝素（PHA）激活的T细胞作为阳性对照，用空白载体pcDNA3.0转染的T细胞作为阴性对照。

结果

生成了针对CEA(+)胃癌细胞的带有嵌合受体的T淋巴细胞。这些T细胞与HCG - 27细胞共培养12小时和24小时后，CD69表达率分别为40.5%±3.4%和48.3%±2.8%。然而，用空白载体转染的T淋巴细胞转染的HGC - 27和SGC - 7901细胞的CD69表达率均为0%。FCM显示，CEA(+)胃肿瘤HGC - 27细胞的凋亡率为47.8%±4.2%，显著高于CEA(-)胃肿瘤SGC - 7901细胞（18.7%±2.8%，P < 0.05）。