用BrdU作探针检测RA对HL-60细胞的诱导分化作用

DETECTION OF RETINOID ACtD INDUCED DIFFERENTIATION OF HUMAN PROMYELOCYTE CELL HL-60 WITH BrdU PROBE

用溴脱氧尿嘧啶核苷(Erdu)作探针标记人早幼粒白血病细胞株HL-60,并用抗Brdu的单克隆抗体(Anti-Brdu)和免疫组化法追踪观察HL-60细胞在全反式维甲酸(RetinoiolAcid,RA)作用下走向分化成熟的可能性。实验发现,经RA处理前,Brdu标记细胞均为早幼粒细胞,经RA处理5天后,Brdu标记细胞中有21％以上分化成熟为杆状核,分叶核粒细胞。由于这些细胞已处于终末分化期,不再进行DNA合成,故细胞中的Brdu是在细胞分化早期掺入的,从而直接证明HL-60细胞可在RA作用下走向分化成熟。

This experiment was designed to obtain direct evidence of human promyelocytc cell line HL-60 differentiation induced by retinoid acid (RA). Exponential growing cells were exposed to Brdu for 6 hours and then were treated with RA for 5 days. As a thymidine analogue, BrdU was incorporated into S-phase cells, the distribution and percentage of BrdU in various kinds of cell nucleus were detected by immunoh-istochemistry with anti-BrdU monoclonal antibody. The results showed that all the BrdU-labeled cells were promyelocyte in the RA-untreated cells. After treated with RA for 5 days, more that 21% Brdu-labeled cells were differentiated into bandshaped and polymorphonuclear cells.Since these cells were at terminal differentiation and were not able to synthesize DNA, the detected BrdU in their nuclei was derived from unmature labeled S-phase cells. Moreover, the nitroblue tetrazolium(NBT) reducing ability of the RA treated cells was increased when compared to that of the untreated cells. The growth curves of the RA treated cells further suggest that RA inhibits HL-60 cell growth. In summary, this experiiment provides evidence in vitro of differentiation of BrdU labeled cells into mature granulocytes RA not only induces differentiation, but also exerts inhibitory effect to the growth of HL-60 cells