人白血病细胞株重组激活基因表达及T细胞受体基因重排的检测(英文)  

Detection of recombination-activating gene expression and T cell receptor gene recombination in human T cell leukemia cell lines

作　　者：邹红云[1] 马骊[1] 罗微[1] 王小宁[2] 

机构地区：[1]南方医科大学生物技术学院分子免疫研究所,广东省广州市510515 [2]华南理工大学生物科学与工程学院,广东省广州市510641

出　　处：《中国临床康复》2006年第25期173-177,共5页Chinese Journal of Clinical Rehabilitation

基　　金：国家重点基础研究发展计划("九七三"计划)(2001CB510008)~~

摘　　要：背景:淋巴细胞特异性重组激活基因编码的重组激活基因1与重组激活基因2蛋白是参与V(D)J重排机制的重要的重组酶。除参与V(D)J重排以外,近年的研究结果表明重组激活基因介导的转位作用可能与染色体易位及淋巴性恶性肿瘤的发生有关,但迄今尚未有明确定论。目的:检测重组激活基因、DNA修复因子Ku70/Ku80和末端脱氧核苷转移酶mRNA表达以及T细胞受体基因重排在人白血病和淋巴瘤细胞株的发生情况。设计:重复测量实验。单位:南方医科大学生物技术学院分子免疫研究所。材料:T淋巴白血病细胞株Jurkat和6T-CEM购自上海细胞生物研究所;T淋巴白血病细胞株Molt-4,皮肤T细胞淋巴瘤细胞株HuT102,Burkitt’s淋巴瘤细胞株Raji和Daudi以及原髓细胞白血病细胞株HL-60和慢性髓原白血病细胞株K562均由本实验室保存。细胞用含有体积分数0.1胎牛血清的RPMI1640培养基于37℃,体积分数0.05CO2条件下培养。方法:实验于2005-10/2006-01在南方医科大学生物技术学院分子免疫研究所完成。采用反转录聚合酶链反应检测重组激活基因1,重组激活基因2,非同源末端连接装置途径中的DNA修复因子Ku70/Ku80,以及末端脱氧核苷转移酶mRNA表达;采用巢式、半巢式聚合酶链反应、连接介导的聚合酶链反应等方法检测T细胞受体重排删除DNA环和T细胞受体β链重组信号序列两端的断裂点。了解参与V(D)J重排过程的基因表达和T细胞受体基因重排中间体的产生情况。主要观察指标:重组激活基因、DNA修复因子Ku70/Ku80和末端脱氧核苷转移酶mRNA表达以及T细胞受体基因重排在人白血病和淋巴瘤细胞株的发生情况。结果:反转录聚合酶链反应检测结果显示:重组激活基因1mRNA在4种T细胞株中均被检测到,在两种B细胞株和两种髓性白血病细胞株中未检测到;重组激活基因2和末端脱氧核苷转移酶mRNA表达仅在Jurkat,Molt-4和6T-BACKGROUND： The lymphocyte-specific recombination-activating gene （RAG） 1 and 2 are essential for initiating V （D）J recombination events in beth T and B cells. In addition to initiating V（D）J recombination, RAG-mediated transposition has also been proposed to contribute to chromosomal translocations and lymphoid malignancy, but the mechanism underlying this activity remains poorly understood. OBJECTIVE： To investigate RAG gene,DNA repair factors Ku70/Ku80 and terminal deoxynucleotidyl transferase（TdT）mRNA expression and T cell recepto（TCR） gene recombination in human leukemia and lymphoma cell lines. DESIGN： Repeated-determined experiment. SETTING： Institute of Molecular Immunology, College of Biotechnology, Southern Medical University. MATERIALS： Four human T leukemia and lymphoma cell lines （Jurkat, Moh-4, HUT-102, 6T-CEM） ,two Burkitt＇s lymphoma（Raji and Daudi） and two myelogenous leukemia cell lines （HL-60 , K-562） were cultured in complete growth medium （Hyclone, USA）, penicillin/streptomycin（50 U/mL and 50 mg/L） at 37 ℃ in a humidified atmosphere in 0.05 volume fraction of CO2. Jurkat and 6T-CEM were bought from SIBS,CAS（Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences）. The other cell lines were conserved by Institute of Molecular Immunology, College of Biotechnology, Southern Medical University. METHODS： The experiment was carried out in Institute of Molecular Immunology, College of Biotechnology, Southern Medical University from October 2005 to January 2006.Reverse transcription-polymerase chain reaction （RT-PCR）was performed to examine the expression of RAG1,RAG2, terminal deoxynucleotidyl transferase（TdT）, Ku70 and Ku80 genes,reparative factors,in pathway of non-homologous end joining （NHEJ-）;Nested and semi-nested PCR reactions were performed with locus-specific primer sets to determine TCR Dβ-Jβ signal joint T-cell receptor excision DNA circles （sjTRECs）; Li

关 键 词：DNA/遗传学 基因重排 白血病 

分 类 号：R733.7[医药卫生—肿瘤]