髓系白血病干细胞培养及其特征鉴定

Culture and identification of myeloid leukemia stem cells

目的对急性髓系白血病(AML)骨髓单个核细胞进行体外培养,获得白血病干细胞并进行特征鉴定。方法体外培养AML患者的骨髓单个核细胞,观察其形态学特征、长期存活及增殖特性,用染色体分析和流式细胞分析技术研究其核型和细胞表面标志,用RT-PCR技术分析其nucleostemin、Bmi-1、ABCG2、prominin1、OCT4、Nanog等干细胞相关基因的表达。结果培养的白血病细胞呈集落样悬浮生长,在体外能存活12个月以上并保持增殖特性。染色体核型呈亚四倍体。表达上述干细胞相关基因。细胞表面表达CD38、CD29、CD44和HLA-DR,弱表达CD19和CD71,不表达CD34、CD13、CD2和CD105。结论经培养获得的白血病细胞,具备肿瘤干细胞的一些特征,提示为白血病干细胞。

Objective To isolate leukemia stem cells in cultured mononuclear cells which were derived from bone marrow of a patient with acute myeloid leukemia （AML） and identify their biological characteristics. Methods The mononuclear cells derived from the bone marrow of the leukemic patient were cultured in vitro. Morphological characteristics, long-term surviving and proliferative charac-teristics were observed. The karyotype and surface markers of the cells were investigated by chromosome analysis and flow cytometry. The expressions of nucleostemin, Bmi-1, ABCG2, prominin-1, OCT4 and Nanog in the cells were detected by RT-PCR. Results The cells growing in both suspended and agminated mode could survived over 12 months and proliferated constantly in vitro. Chromosomal karyotype of the cells was hypotetraploid. The genes of nucleostemin, Bmi-1, ABCG2, prominin-1, OCT4 and Nanog were all ex- pressed in the cells. The surface markers on the cells were positive for CD38, CD29, CD44 and HLA-DR, positive weakly for CD19 and CD71, but negative for CD34, CD13, CD2 and CD105. Conclusion The leukemic cells obtained from the cultured mononuclear cells exhibited some characteristics of cancer stem cell, which indicates the cells may be leukemia stem cells.