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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-CEM3种T细胞株中检测到,但在6T-CEM表达较弱;除HL-60细胞未检测到Ku80表达外,所有细胞株均检测到Ku70和Ku80表达。对4种T细胞株T细胞受体重排中间体检测结果表明:仅在Jurkat细胞中检测到Dβ2-Jβ2sjTRECs与Dβ25’端和3’RSS断点,表明Jurkat细胞发生T细胞受体基因重排。同时发现JurkatTCRDβ2-Jβ2重排删除环结合区具有明显的多样性特征。结论:重组激活基因可能与T细胞白血病具有更为密切的关系。Jurkat细胞有可能成为研究重组激活基因与T细胞淋巴性肿瘤的一个潜在的细胞模型。
BACKGROUND: Recombinant activator gene 1 and recombinant activator protein 2 encoded by lymphocyte-specific recombinant activator genes are important recombinases involved in the V (D) J rearrangement mechanism. In addition to participating in V (D) J rearrangement, recent studies have shown that the transactivation mediated by recombinant activated genes may be related to the occurrence of chromosomal translocations and lymphoid malignancies, but so far no definite conclusion has been reached. OBJECTIVE: To detect the expression of recombinant activation genes, DNA repair factor Ku70 / Ku80 and terminal deoxynucleotidyl transferase mRNA and the gene rearrangement of T cell receptors in human leukemia and lymphoma cell lines. Design: Repeat measurement experiment. Unit: Institute of Molecular Immunology, College of Biotechnology, Southern Medical University. MATERIALS: T lymphoid leukemia cell lines Jurkat and 6T-CEM were purchased from Shanghai Institute of Cell Biology; Molt-4 cell lines of T lymphoid leukemia, HuT102 of cutaneous T-cell lymphoma, Raji and Daudi of Burkitt’s lymphoma cell lines, Leukemia cell line HL-60 and chronic myeloid leukemia cell line K562 were preserved in this laboratory. The cells were cultured in RPMI 1640 medium containing 0.1 volume fraction of fetal bovine serum at 37 ° C under a volume fraction of 0.05 CO2. Methods: The experiment was performed at Institute of Molecular Immunology, School of Biotechnology, Southern Medical University from October 2005 to January 2006. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of recombinant activation gene 1, recombinant activation gene 2, DNA repair factor Ku70 / Ku80, and terminal deoxynucleotidyl transferase mRNA in the pathway of non-homologous end- Nested polymerase chain reaction, ligation-mediated polymerase chain reaction and other methods to detect T cell receptor rearrangement delete DNA loop and T cell receptor β chain recombination signal sequence at both ends of the breakpoint. To understand the gene expression involved in V (D) J rearrangement and the production of T cell receptor gene rearrangement intermediates. MAIN OUTCOME MEASURES: The expression of recombinant activated genes, DNA repair factor Ku70 / Ku80 and terminal deoxynucleotidyltransferase mRNA, and T cell receptor gene rearrangements in human leukemia and lymphoma cell lines. Results: The results of RT-PCR showed that the recombinant activated gene 1 mRNA was detected in all four T cell lines and not detected in the two B cell lines and the two myeloid leukemia cell lines. Recombinant activation Gene 2 and terminal deoxynucleotidyltransferase mRNA expression were detected only in 3 T cell lines, Jurkat, Molt-4 and 6T-CEM, but weakly expressed in 6T-CEM; except Ku80 expression was not detected in HL-60 cells , Ku70 and Ku80 expression were detected in all cell lines. T cell receptor rearrangement intermediates of four T cell lines showed that Dβ2-Jβ2sjTRECs and Dβ25 ’end and 3’RSS breakpoint were detected only in Jurkat cells, indicating that T cell receptor gene rearrangement occurred in Jurkat cells . At the same time, JurkatTCRDβ2-Jβ2 rearrangement and deletion of the loop binding region showed obvious diversity characteristics. Conclusion: Recombinant activator may play a more important role in T cell leukemia. Jurkat cells may serve as a potential cellular model for the study of recombinant activated genes and T-cell lymphomas.