Hua J, Hasebe T, Someya A, Nakamura S, Sugimoto K, Nagaoka I
Department of Biochemistry, Juntendo University, School of Medicine, Tokyo, Japan.
J Leukoc Biol. 2000 Aug;68(2):216-24.
To understand the expression of NADPH oxidase components during neutrophil maturation, we examined the expression of mRNAs and proteins for NADPH oxidase components, and the superoxide-producing activity using HL-60 cells incubated with dimethyl sulfoxide (DMSO). Northern blot and Western blot analyses revealed that gp91(phox), p67(phox), and p47(phox) were expressed after myelocyte stages, whereas p22(phox), p40(phox), and rac-2 were expressed from the promyelocyte stage. Furthermore, immunocytochemical staining of DMSO-induced HL-60 cells indicated that gp91(phox), p67(phox), and p47(phox) were detected only after myelocyte stages (myelocytes, metamyelocytes, band cells, and segmented cells), whereas p22(phox), p40(phox), and rac-2 were detected from the promyelocyte stage. In addition, nitro blue tetrazolium (NBT) assay showed that superoxide could be produced after myelocyte stages but not produced before promyelocyte stages. Moreover, almost the same results as those with DMSO-induced HL-60 cells were obtained using human bone-marrow cells by immunocytochemical staining and NBT assay, except that p22(phox) was detected by immunocytochemical staining after myelocyte stages in bone-marrow cells. Together, these observations indicate that all the components for NADPH oxidase are expressed, and the superoxide-producing activity is obtained after myelocyte stages during neutrophil maturation.
为了解中性粒细胞成熟过程中NADPH氧化酶各组分的表达情况，我们检测了用二甲基亚砜（DMSO）孵育的HL-60细胞中NADPH氧化酶各组分的mRNA和蛋白表达，以及超氧化物生成活性。Northern印迹和Western印迹分析显示，gp91(phox)、p67(phox)和p47(phox)在中幼粒细胞阶段之后表达，而p22(phox)、p40(phox)和rac-2从早幼粒细胞阶段开始表达。此外，DMSO诱导的HL-60细胞的免疫细胞化学染色表明，gp91(phox)、p67(phox)和p47(phox)仅在中幼粒细胞阶段（中幼粒细胞、晚幼粒细胞、杆状核细胞和分叶核细胞）之后才能检测到，而p22(phox)、p40(phox)和rac-2从早幼粒细胞阶段就能检测到。另外，硝基蓝四氮唑（NBT）试验表明，超氧化物可在中幼粒细胞阶段之后产生，但在早幼粒细胞阶段之前不产生。此外，通过免疫细胞化学染色和NBT试验，用人骨髓细胞获得了与DMSO诱导的HL-60细胞几乎相同的结果，不同之处在于骨髓细胞中，p22(phox)在中幼粒细胞阶段之后通过免疫细胞化学染色才能检测到。总之，这些观察结果表明，NADPH氧化酶的所有组分均有表达，并且在中性粒细胞成熟过程中，超氧化物生成活性在中幼粒细胞阶段之后获得。
