转染NY-ESO-1特异性TCR增强人PBMC对NY-ESO-1阳性肿瘤细胞的特异性细胞毒性  

Transfection of NY-ESO-1 specific TCR improves the specific cytotoxicity of human PBMC to NY-ESO-1 positive tumor cells

作　　者：郭佳[1] 田洲[1] 顾娜[1] 徐珩[2] 闾军[1] 

机构地区：[1]首都医科大学附属北京佑安医院肝病与肿瘤生物治疗科,北京100069 [2]安徽医科大学基础医学院,安徽合肥230032

出　　处：《基础医学与临床》2013年第3期303-307,共5页Basic and Clinical Medicine

基　　金：北京市自然科学基金(7092044);首都医学发展科研基金(2009-3156);北京市卫生系统高层次卫生技术人才培养项目资助(2011-J.L)

摘　　要：目的探讨体外转导NY-ESO-1特异性T细胞受体至人外周血淋巴细胞中,是否可增强转导后细胞体外特异性识别并杀伤肿瘤细胞的能力。方法将NY-ESO-1特异性TCR质粒(pCDNA3.1-ESO-TCR)体外电转入新分离的正常人PBMC中,RT-PCR法鉴定转导是否成功;采用流式细胞仪分析转导后PBMC表型;用特异性NY-ESO-1b抗原肽(p157-165)刺激转导PBMC后,用ELISPOT法检测PBMC分泌IFN-γ的能力,用实时无标记动态细胞分析仪检测PBMC特异性细胞毒性作用。结果电转后PBMC能够扩增出特异性TCR片段;电转pCDNA3.1-ESO-TCR质粒的PBMC细胞表面特异性TCR表达率高于电转空载体的PBMC的表达率(P<0.05);经多肽刺激后,电转目的质粒的PBMC分泌IFN-γ的斑点数明显多于电转空载体的PBMC(P<0.05);转导后PBMC特异性细胞毒性作用明显强于电转空载体的PBMC。结论经NY-ESO-1特异性TCR基因修饰的PBMC对NY-ESO-1阳性HepG2细胞的体外特异性杀伤作用有明显提高,为进行下一步肝癌过继性免疫研究提供了基础资料。Objective To study whether NY-ESO-l-specific TCR gene transduced into human PBMC （Peripheral Blood Mononuclear Cell, PBMC） can improve its abilities of specifically recognizing and killing tumor cells. Methods pCDNA3. 1-ESO-TCR plasmid was electroporately transferred into PBMC separated from healthy people in vitro and confirmed by RT-PCR. The phenotype analysis after eletroporation was measured by flow cytometry meth- od. The NY-ESO-1 specific peptide （p157-165） was added into the culture of electroporated PBMC, the IFN-y/ level secreted by electroporated PBMC was detected by ELISPOT assay. The specific cytotoxicity in vitro was detected by real time cell analysis（ACEA biosciences）. Results The NY-ESO-l-specific TCR fragments in electroporatedPBMC were detected by RT-PCR. The specific expression of NY-ESO-TCR in transferred PBMC was significantly higher than that in PBMC transfered with empty vector （ P 〈 0. 05 ）. The positive dots of IFN-y secretion in trans- ferred PBMC stimulated by peptide p157-165 was significantly more than that in PBMC transfered with empty vector （ P 〈0. 05） ;The specific cytotoxicity of transferred PBMC was obviously enhanced than PBMC transduced with empty vector. Conclusions The specific cytotoxicity of PBMC against NY-ESO-1 positive HepG2 cells is elevated by transduced PBMC with NY-ESO-1 specific TCR, which may stimulate more researches about adoptive immunotherapy of hepatocellular carcinoma.

关 键 词：TCR PBMC 电转 NY-ESO-1 

分 类 号：R730.2[医药卫生—肿瘤]