TCR Vα23-Vβ13基因修饰T细胞及其特异性细胞毒活性研究  

Specific cytotoxicity of TCR Vα23-Vβ13 gene-modified T-cells

作　　者：尹青松[1,2] 谭获[3] 杨力建 陈少华 查显丰 叶静梅[3] 李扬秋[2,4] 

机构地区：[1]河南省肿瘤医院,河南郑州450008 [2]医学院血液病研究所 [3]广州医学院第一附属医院肿瘤血液中心,广东广州510260 [4]再生医学教育部重点实验室,广东广州510632

出　　处：《中国病理生理杂志》2011年第2期260-267,共8页Chinese Journal of Pathophysiology

基　　金：国家高技术研究发展计划资助项目(No.2006AA02Z114);广东自然科学基金重点资助项目(No.05103293;No.9251063201000001)

摘　　要：目的:分析弥漫大B细胞淋巴瘤(DLBCL)抗原特异T细胞受体(TCR)Vα23-Vβ13基因修饰T细胞后的体外特异性杀伤活性。方法:扩增并克隆已鉴定的DLBCL患者外周血中单克隆增殖T细胞的TCR Vβ13和TCR Vα23基因全长序列,构建TCR Vβ13-IRES-TCR-Vα23双基因重组质粒,经核转染的方法将其转染正常人T细胞,通过real-time PCR和Western blotting检测TCR Vβ13和TCR Vα23这2个外源基因在mRNA和蛋白水平的体外表达情况,并对TCR转基因T细胞进行体外细胞毒性检测。结果:经酶切分析和核酸序列测定证实重组质粒构建正确,转染正常人T细胞后,TCR Vβ13和TCR Vα23在mRNA及蛋白水平实现了共表达。转染后3 d,TCR转基因T细胞对DLBCL细胞株Toledo细胞的杀伤活性明显高于非特异细胞株Raji细胞和Molt-4细胞,显示出特异性细胞毒活性。结论:成功构建DLBCL相关抗原特异的TCR Vβ13-IRES-TCR Vα23双基因真核表达质粒;TCR基因修饰T细胞可获得特异性细胞毒活性。AIM ： To analyze the cytotoxicity of TCR Vα23 - Vβ13 gene - modified T - cells specific to diffuse large B- cell lymphoma（DLBCL） associated antigens in vitro. METHODS： The identified full -length TCR Vβ13 and Vα23 genes of monoclonally expanded T - cells in the peripheral blood from a DLBCL patient were amplified and cloned. The bicistronic recombinant plasmid TCR Vβ13 - IRES - TCR Vα23 was constructed and transfected into T - cells isolated from a healthy individual by NucleofectorTM technology. The mRNA expression of antigen - specific TCR Vα23 and Vβ13 genes and the corresponding proteins were determined by real -time PCR and Western blotting, respectively. The specific cytotoxicity of TCR gene - transferred T - cells in vitro was detected. RESULTS ： The recombinant plasmid was constructed successfully and verified by restriction enzyme digestion and nucleotide sequencing. The co - expression of antigen - specific TCR Vα23 and Vβ13 genes at mRNA and protein levels in the transfected healthy T - cells were observed in vitro. Three days after transfection, the cytotoxicity of TCR gene - transduced CD3 ＋ T - cells against Toledo ceils was significantly higher than that against Raji cells or Molt - 4 cells. The DLBCL - specific cytotoxic T - lymphocytes（CTL） were inducted. CONCLUSION ： The bicistronic eukaryotic expression plasmid TCR Vβ13 - IRES - TCR Vα23 specific to DLBCL - asso- ciated antigens is constructed successfully. The TCR gene - transferred T - cells display the ability of DLBCL - specific cytotoxicity.

关 键 词：弥漫大B细胞淋巴瘤 TCR转基因 细胞毒活性 

分 类 号：R392.11[医药卫生—免疫学] R733.4[医药卫生—基础医学]