NY-ESO-1特异性TCR转导人外周血淋巴细胞及相关检测

基础医学与临床 ›› 2013, Vol. 33 ›› Issue (3): 303-307.

NY-ESO-1特异性TCR转导人外周血淋巴细胞及相关检测

郭佳¹,田洲¹,顾娜¹,徐珩²,闾军¹

1. 首都医科大学附属北京佑安医院
2. 安徽医科大学

收稿日期:2012-11-06 修回日期:2013-01-17 出版日期:2013-03-05 发布日期:2013-03-05
通讯作者: 闾军 E-mail:lujun98@gmail.com
基金资助:
北京市自然科学基金

Functional analysis of the lymphocytes transduced with NY-ESO-1 specific T cell receptor

Received:2012-11-06 Revised:2013-01-17 Online:2013-03-05 Published:2013-03-05

摘要/Abstract

摘要： 目的：将具有肿瘤抗原NY-ESO-1特异性的T细胞受体(T Cell Receptor，TCR)转导至人外周血淋巴细胞(Peripheral Blood Lymphocytes，PBLs)中，提高转导后细胞表面特异性TCR表达效率，特异性增强转导后细胞识别肿瘤抗原及分泌IFN-γ的能力，为下一步进行肿瘤杀伤实验奠定基础。方法：将实验室保存的NY-ESO-1特异性T细胞受体质粒（pCDNA3.1-ESO-TCR）体外电转入新分离的正常人PBLs中，RT-PCR法鉴定转导是否成功；通过流式细胞仪对转导后的PBLs细胞进行表型分析；用特异性NY-ESO-1b抗原肽 (p157-165) 刺激转导阳性的PBLs，ELISPOT法检测转导后PBLs细胞分泌IFN-γ的能力。结果：RT-PCR检测到电转后PBLs能够扩增出特异性TCR片段；流式检测结果显示电转pCDNA3.1-ESO-TCR质粒的PBLs细胞表面特异性TCR表达率高于未电转质粒的PBLs的表达率(P<0.05)；经多肽刺激后，与未电转质粒的PBLs相比，电转质粒的PBLs分泌IFN-γ的斑点数明显增多(P<0.05)。结论：通过体外转导的方式提高NY-ESO-1特异性TCR 在PBLs表面的表达，能够特异性增强PBLs分泌IFN-γ的效率，为下一步进行肿瘤杀伤实验奠定基础。

关键词: TCR, PBL, 电转, NY-ESO-1

Abstract: Objective: NY-ESO-1-specific TCR gene was transduced into human peripheral blood lymphocytes (PBLs) to improve its ability of specifically recognizing and killing tumor cells. Method: pCDNA3.1-ESO-TCR plasmid was electroporately transferred into PBLs separated from healthy people in vitro and confirmed by RT-PCR. The phenotype analysis after eletroporation was measured by flow cytometry method. The NY-ESO-1 specific peptide (p157-165) was added in the culture of electroporated PBLs, the IFN-γ level secreted by electroporated PBLs was detected by ELISPOT assay. Result: The NY-ESO-1-specific TCR fragments in electroporated PBLs were detected by RT-PCR. The specific expression of NY-ESO-TCR in transferred PBLs is significantly higher than that in untransferred PBLs (P<0.05). The positive dots of IFN-γ secretion in transferred PBLs stimulated by peptide p157-165 was significantly more than that in untransferred PBLs (P<0.05). Conclusion: The NY-ESO-1 specific TCR expression in PBLs was elevated by optimized electroporation, which could improve the efficiency of IFN-γ secretion in human peripheral blood lymphocytes and provide the possibility for cancer immunotherapy.

Key words: TCR, PBLs, Electroporation, NY-ESO-1

中图分类号:

R322.4

郭佳田洲顾娜徐珩闾军. NY-ESO-1特异性TCR转导人外周血淋巴细胞及相关检测[J]. 基础医学与临床, 2013, 33(3): 303-307.

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