白血病K562细胞株中CD34^+细胞群的分离及其生物学特性

Isolation and biological characteristics of CD34^+ cell population from leukemia K562 cell strain

目的分离白血病K562细胞株中CD34+细胞群,并分析其生物学特征,为从干细胞角度治疗白血病提供实验依据。方法采用免疫磁性分选法分离K562细胞中CD34+细胞群,台盼蓝拒染法检测细胞活性;流式细胞术检测CD34+细胞比例及细胞周期;单细胞克隆培养检测CD34+细胞自我更新能力;RT-PCR法检测CD34+细胞分化相关指标促红细胞生成素(Erythropoietin,EPO)和粒细胞-巨噬细胞集落刺激因子(Granulocyte macrophage colony stimulatingfactor,GM-CSF)基因mRNA的转录水平;并检测CD34+细胞耐药蛋白P-gp(P-glycoprotein)的表达。结果免疫磁性分选法可有效分离出CD34+细胞群,细胞活性为99%～100%;分离的CD34+细胞含量占细胞总数的78.5%～85.3%,细胞大部分处于静止状态,G0/G1期细胞比例达80%左右,显著高于分离前的K562细胞;CD34+细胞群具有形成混合集落的能力;与K562细胞相比,CD34+细胞EPO和GM-CSF基因mRNA的转录水平明显下降(P<0.01),P-gp表达阳性。结论成功从K562细胞株中分离了CD34+细胞群,其具有自我更新和多向分化的能力,表明其具有白血病干/祖细胞的生物学特点。

Objective To isolate CD34＋ cell population from leukemia K562 cell line,analyze its biological characteris-tics and provide an experimental basis for stem cells-based treatment of leukemia.Methods CD34＋ cell population was isolated from K562 cells by magnetic cell sorting（MACS）,and determined for activity by trypan blue dye exclusion method,for percentage and cell cycle of CD34＋ cells by flow cytometry,for self-renewal ability by single cell clone culture,and for transcription levels of erythropoietin（EPO） and granulocyte macrophage colony stimulating factor（GM-CSF） mRNAs by RT-PCR.Meanwhile,the expression of P-glycoprotein（P-gp） in CD34＋ cell population was determined.Results CD34＋ cell population was effectively isolated by MACS,of which the activity was 99% ～ 100%.The isolated CD34＋ cells accounted for 78.5% ～ 85.3% of total cells,of which the percentage at phases G0 / G1 was about 80%,significantly higher than that of K562 cells before MACS.The CD34＋ cells showed ability in formation of colonies,in which the transcription levels of EPO and GM-CSF mRNAs decreased significantly as compared with those in K562 cells（P 0.01）,while P-gp was expressed.Conclusion CD34＋ cell population was successfully isolated from K562 cell strain,which showed abilities in self-renewal and multi-directional differentiation.