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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 human acute monocytic leukemia (AMLM5b) cell line and study its biological characteristics. Methods Mononuclear cells were isolated from bone marrow samples of 1 AMLM5b patients with relapsed leukemia and cultured with liquid culture method. The cells were stained with Reiter stain, electron microscopy, cytochemical staining, flow cytometry, R-banding karyotyping, reverse transcriptase polymerase chain reaction (RTPCR), fluorescence in situ hybridization , Nude mice tumorigenicity experiment, fluorescence quantitative PCR, DNA fluorescence staining and mycoplasma broth culture, short tandem repeat (STR) PCR, p53 gene PCR amplification product sequencing, multicolor FISH (MFISH) and 3HTdR doping Into experiments and other methods to identify the biological characteristics of SHI1 cells. Results A monoliferative human monocytic leukemia cell line, SHI1, was established. Morphology and immunophenotype showed typical characteristics of mononuclear cells. Karyotype analysis showed that the SHI1 cell line had the same abnormalities as the bone marrow cells The transcripts of MLLAF6 fusion gene were detected by RTPCR. The results of FISH showed that there was a translocation between chromosomes 6 and 11, MLL gene rearrangement and p53 gene deletion; PCR product sequencing results showed that a p53 allele on the 6th exon point mutation ATC → ACC colony culture showed SHI1 cells have strong colony forming ability; subcutaneous 4 inoculum Mice formed solid tumors. Fluorescence quantitative PCR showed no EBV infection. Mycoplasma was not detected by DNA fluorescence staining and Mycobacterium broth culture. MFISH confirmed that all the SHI1 cells passaged to March 2003 except t (6; 11) , Del (17) (p11),?