Matsuhisa T, Mori Y
Department of Cell Biology, Center for Adult Diseases, Osaka, Japan.
Blood Cells Mol Dis. 1995;21(1):42-8. doi: 10.1006/bcmd.1995.0007.
Differentiation of human promyelocytic leukemia cells, HL-60, has been extensively studied. In this study we utilized 1 alpha,25-dihydroxyvitamin D3 (D3) as a potent inducer and cellular superoxide production as the functional differentiation marker. We examined how nitroblue tetrazolium (NBT) positive cells were produced in the presence of D3. Growth of HL-60 cells tended to level off when the curve was drawn on a logarithmic scale but they grew linearly on a normal scale. When absolute numbers of NBT positive or negative cells were plotted, NBT positive cells only increased linearly in a normal scale, whereas NBT negative cells remained constant after they doubled themselves. When cells were sparsely inoculated in 0.3% agar and cultured for 4 days in the presence of D3, clusters of cells were stained with NBT. Each cluster of cells was composed of one or two NBT negative and three to six NBT positive cells. After treatment with nocodazole and D3, cells were cultured further in the presence of D3. It was demonstrated that only NBT positive cells increased abruptly. Based on these results, differentiated cells might be produced and accumulate through mitosis. In the presence of D3 NBT negative cells remained constant in number and continued to produce NBT positive cells, working as so-called stem cells.
人早幼粒细胞白血病细胞HL-60的分化已得到广泛研究。在本研究中，我们使用1α,25-二羟基维生素D3（D3）作为强效诱导剂，并将细胞超氧化物生成作为功能分化标志物。我们研究了在D3存在的情况下如何产生硝基蓝四氮唑（NBT）阳性细胞。当以对数尺度绘制曲线时，HL-60细胞的生长趋于平稳，但在正常尺度上它们呈线性生长。当绘制NBT阳性或阴性细胞的绝对数量时，NBT阳性细胞仅在正常尺度上呈线性增加，而NBT阴性细胞在自身翻倍后保持恒定。当将细胞稀疏接种于0.3%琼脂中并在D3存在下培养4天时，细胞簇被NBT染色。每个细胞簇由一两个NBT阴性细胞和三到六个NBT阳性细胞组成。用诺考达唑和D3处理后，细胞在D3存在下进一步培养。结果表明，只有NBT阳性细胞急剧增加。基于这些结果，分化细胞可能通过有丝分裂产生并积累。在D3存在的情况下，NBT阴性细胞数量保持恒定，并继续产生NBT阳性细胞，起到所谓干细胞的作用。
