一株伴有t(6;11)(q27;q23)和p53基因异常的人单核细胞白血病细胞系SHI-1的建立及鉴定  

Establishment and characterization of a human acute monocytic leukemic cell line, SHI-1, carrying t(6;11)(q27;q23) and p53 gene alteration

作　　者：陈苏宁[1] 薛永权[1] 张学光[2] 吴亚芳[1] 潘金兰[1] 王勇[1] 岑建农[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所,215006 [2]苏州大学生物技术研究所

出　　处：《中华血液学杂志》2005年第2期94-99,共6页Chinese Journal of Hematology

基　　金：国家863计划资助项目(X19010105);国家自然科学基金面上项目(30370596);江苏省135重点学科开放课题基金资助项目(135XY4014);苏州市科技项目(ZS0201)

摘　　要：目的建立人急性单核细胞白血病(AMLM5b)细胞系并研究其生物学特性。方法从1例AMLM5b患者白血病复发时的骨髓标本分离出单个核细胞,用液体培养法进行培养。采用瑞特染色、电子显微镜、细胞化学染色、流式细胞仪、R显带核型分析、逆转录聚合酶链反应(RTPCR)、荧光原位杂交(FISH)、半固体甲基纤维素集落培养、裸小鼠致瘤实验、荧光定量PCR、DNA荧光染色法及支原体肉汤培养法、短串联重复序列(STR)PCR、p53基因的PCR扩增产物测序、多色FISH(MFISH)和3HTdR掺入实验等方法对SHI1细胞的生物学特性进行了鉴定。结果建立了1个可持续增殖的人单核细胞白血病细胞系SHI1;形态学和免疫表型呈现典型的单核系特征;核型分析显示SHI1细胞系有和患者复发时骨髓细胞完全相同的异常46,XY,t(6;11)(q27;q23),del(17)(p11);RTPCR检出MLLAF6融合基因的转录本;FISH检测结果显示存在6号和11号染色体之间易位、MLL基因的重排和p53基因的缺失;PCR产物测序结果显示1个p53等位基因6号外显子发生点突变ATC→ACC集落培养显示SHI1细胞具有较强的集落形成能力;皮下接种4只裸小鼠均形成实体肿瘤;荧光定量PCR提示无EB病毒感染;DNA荧光染色法和支原体肉汤培养法未检出支原体;MFISH证实传代至2003年3月的SHI1细胞除有t(6;11)、del(17)(p11)外,?Objective To establish a novel human monocytic leukemic cell line and characterize its biological features. Methods Mononuclear cells isolated from the bone marrow of an acute monocytic leukemia (AML-M_~5b ) patient at relapse were inoculated in a liquid culture system. And the biologic features of the established cell line SHI-1 were characterized by morphology, cytochemical staining, flow cytometry, karyotypic analysis, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), fluorescence in situ hybridization (FISH), tumorigenicity in nude mice, quantitative fluorescent polymerase chain reaction, broth medium culture, short tandem repeating sequences-PCR (STR-PCR), multiplex-FISH (M-FISH), and ~3 H-thymidine incorporation assay. Results A human acute monocytic leukemia cell line, SHI-1, was established and has proliferated continuously in vitro for over one year. The cell line presented typical morphology and immunoprofile of monocytic lineage with the original t(6;11)(q27;q23) and del(17)(p11) abnormalities. The MLL-AF6 fusion transcript was detected by RT-PCR. The rearrangement of MLL gene, deletion of p53 gene, and translocation between chromosomes 6 and 11 were revealed by FISH. A point mutation of ATC→ACC at exon 6 of the p53 gene was found by sequencing of the PCR products. The clonality and the high tumorigenicity of the SHI-1 cell line were confirmed. Infections of EBV and mycoplasma were excluded. A derivative chromosome 7 resulting from a translocation between chromosomes 7 and 13, monosomy 18 and a minute derived from chromosome 8 in addition to t(6;11) and deletion(17)(p11) were detected by M-FISH in SHI-1 cells passaged to March 2003. Cell line authentication by STR-PCR confirmed the identity to the original leukemic cells of the patient. ~3 H-thymidine incorporation assay showed that IL-4 and IL-15 had proliferative effects, while IFN-γ, TNFα, IL-2, PDGF, and IL-7 had inhibitory effects on the cell line . ConclusionsSHI-1 is a novel acute monocytic leukemia-derived cell line carry

关 键 词：SHI 人单核细胞 P53基因 白血病细胞系 患者 裸小鼠 增殖 短串联重复序列(STR) 显带 PCR产物 

分 类 号：R733.7[医药卫生—肿瘤]