Wallington L A, Durham J, Bunce C M, Brown G
Department of Immunology, Medical School, University of Birmingham, UK.
Leuk Res. 1996 Oct;20(10):821-9. doi: 10.1016/s0145-2126(96)00040-9.
Treatment of serum-free grown HL60 cells with certain combined amounts of retinoic acid (9-cis or all-trans RA) and 1 alpha 25 dihydroxyvitamin D3 (D3) results in differentiation of 71-77% of cells towards either neutrophils or monocytes. Studies of the differentiation of HL60 cells in flask cultures does not reveal: (i) the extent to which selective growth of cells might have occurred; and (ii) the overall level of cell survival. This information can be obtained by monitoring the effects of differentiative agents on individual cells. Serum-free grown HL60 cells were cultured as single cells in microtitre wells in conditioned medium obtained from exponentially growing and serum-free cultures of HL60. This resulted in a cloning efficiency of 85% and HL60 cells doubled every 24 h. During a period of exponential growth < 0.5 to 2% of the cells generated died. Single HL60 cells were treated with 9-cis and all-trans RA (5 x 10(-7) M) together with a small amount of D3 (3.9 x 10(-14) M) to promote neutrophil differentiation. D3 alone (10(-7) M) and D3 (5 x 10(-9) M) in combination with 9-cis RA (10(-8) M) were used to promote monocyte differentiation. The growth kinetics of HL60 cell cultures that were differentiating to neutrophils and to monocytes were similar. Single-cell experiments have revealed that: (i) differentiating HL60 cells undergo a variable number of divisions (two to five) prior to arresting their growth; and (ii) up to 33% of the cells that are generated (by day 5) die. Seventy to eighty per cent of the cells in each of the wells had matured. These findings have important implications in regard to whether retinoids and D3 provide signals that determine the choice of maturation pathway or that merely facilitate selective survival and/or expansion of cells that have independently determined their differentiation fates.
用特定组合量的视黄酸（9-顺式或全反式视黄酸）和1α,25-二羟基维生素D3（D3）处理无血清培养的HL60细胞，会使71%-77%的细胞分化为中性粒细胞或单核细胞。对培养瓶培养的HL60细胞分化的研究未揭示：（i）细胞选择性生长可能发生的程度；以及（ii）细胞存活的总体水平。通过监测分化剂对单个细胞的影响可获得此信息。将无血清培养的HL60细胞作为单细胞培养在微孔板孔中，所用条件培养基取自指数生长的无血清HL60培养物。这导致克隆效率为85%，HL60细胞每24小时加倍。在指数生长期，产生的细胞中有不到0.5%至2%死亡。用9-顺式和全反式视黄酸（5×10⁻⁷M）以及少量D3（3.9×10⁻¹⁴M）处理单个HL60细胞以促进中性粒细胞分化。单独使用D3（10⁻⁷M）以及D3（5×10⁻⁹M）与9-顺式视黄酸（10⁻⁸M）联合使用以促进单核细胞分化。向中性粒细胞和单核细胞分化的HL60细胞培养物的生长动力学相似。单细胞实验表明：（i）正在分化的HL60细胞在停止生长前经历不同数量的分裂（两到五次）；以及（ii）（到第5天）产生的细胞中多达33%死亡。每个孔中70%至80%的细胞已经成熟。这些发现对于视黄酸和D3是提供决定成熟途径选择的信号，还是仅仅促进已独立确定其分化命运的细胞的选择性存活和/或扩增，具有重要意义。
