免疫磁珠分选白血病KG1a细胞中CD34^+CD38^-干细胞及其特性研究

Isolation and characteristic of CD34^+CD38^- stem cells in leukemia cell line KG1a using magnetic activated cell sorting

目的从白血病KG1a细胞中分选CD34+CD38-干细胞并研究其生物学特性。方法免疫磁珠法分选CD34+CD38-细胞,流式细胞术分析细胞表面膜抗原、细胞周期,甲基纤维素培养体系观察其克隆性;以HL60、K562、CD34+CD38+细胞为对照,甲基偶氮唑蓝法检测柔红霉素对CD34+CD38-细胞的抑制作用;BALB/c裸鼠皮下接种,观察体内成瘤能力。结果分选的CD34+CD38-细胞纯度达95%以上,(69.03±3.25)%处于G0期,克隆形成率为(38.64±2.68)%,明显抵抗柔红霉素;不同浓度柔红霉素作用后,CD34+CD38-、CD34+CD38+、HL60、K562细胞的活性差异有统计学意义(F=961.136,P=0.000);CD34+CD38-在裸鼠皮下成瘤率显著高于CD34+CD38+细胞(P<0.05)。结论免疫磁珠法分选白血病干细胞简单易行,分选的细胞符合白血病干细胞生物学特性。

Objective To isolate the CD34＋CD38-stem cells from human acute leukemia cell line KG1a and to research its biological characteristics.Methods CD34＋CD38-cells were isolated by magnetic activated cell sorting（MACS）;the cell surface membrane antigens and cell cycle were analyzed by flow cytometry;its clonality was observed with methyl cellulose system.HL60,K562 and D34＋CD38＋ cells were selected as control,and the methyl thiazolyl tetrazolium（MTT）assay was taken to observe the depressant effect of rubidomycin to CD34＋CD38-cells.BALB/c nude mice were inoculated subcutaneously to observe the tumorigenicity.Results The purity of CD34＋CD38-cells were above 95% and（69.03±3.25）% cells in the G0 phase.The cloning efficiency of CD34＋CD38-cells were（38.64±2.68）%,and the CD34＋CD38-cells were obviously resistant to rubidomycin.There were statistic difference of cytoactive among CD34＋CD38-,CD34＋CD38＋,HL60,and K562 cells under giving the same concentrations of rubidomycin circumstances（F= 961.136,P= 0.000）.The tumorigenesis ability of CD34＋CD38-cells in nude mice was significantly higher than that of CD34＋CD38＋ cells（P0.05）.Conclusion MACS is a simple method for isolating the leukemic stem cells and the isolated cells are consistent with the biology characteristics of the leukemic stem cells.