红系衍生核因子相关因子2基因在芬维A胺诱导NB4细胞凋亡中的作用

Effects of nuclear factor(erythroid-derived 2)-like 2 on fenretinide induced apoptosis in NB4 cells

目的:研究干扰红系衍生核因子相关因子2(NRF-2)基因表达对芬维A胺诱导NB4白血病细胞凋亡的影响,探寻白血病治疗的新思路。方法:采用核转染技术将NRF-2的小干扰RNA(siRNA)转入NB4细胞干扰NRF-2的表达,然后再用芬维A胺(1μmol/L)处理NB4细胞,24、48 h后应用流式细胞术膜联蛋白(Annexin-V)异硫氰酸荧光素(FITC)/碘化丙啶(PI)双标记法检测细胞凋亡情况。结果:1μmol/L芬维A胺能明显诱导NB4细胞凋亡,并呈时间依赖性和药物剂量依赖性,在凋亡发生过程中NRF-2的mRNA和蛋白水平有上升趋势。通过RNA干扰NRF-2表达后,NRF-2的mRNA以及蛋白水平都明显下降,芬维A胺诱导NB4细胞凋亡的效能也随之减弱。流式细胞术分析干扰组细胞凋亡率仅(29.90±2.45)%,明显低于非干扰组的(43.85±14.40)%(P=0.001)。结论:NRF-2在芬维A胺诱导NB4细胞凋亡过程中扮演着重要的角色,提示芬维A胺可能通过氧化应激途径诱导细胞凋亡。通过这样的途径,设计提高白血病细胞的氧化应激能力可能成为靶向治疗白血病的新途径。

Objective To investigate the effect of interfering the expression of nuclear factor （erythroid-derived 2）- like 2 （NRF-2） on fenretinide induced apoptosis in NB4 leukemia cells, and to explore a new approach for the targeting therapy of leukemia. Methods Nucleotransfection technique was used to tranfect NRF-2 small interfering RNA （siRNA） into NB4 cells to interfere the expression of NRF-2, then the NB4 cells were treated with 1 ~mol/L fenretinide. Reverse transcript polymerase chain reaction （RT-PCR） and Western blotting analysis were used to detect the mRNA and protein levels of NRF-2, and flow cytometry （FCM） was used to measure the apoptosis rate of NB4 cells 24 and 48 hours after treated with fenretinide. Results Fenretinide at a concentration of 1 Ixmol/L could obviously induce the apoptosis of NB4 cells in a time and dose dependent manner, and the mRNA and protein levels of NRF-2 were up-regulated during the apoptosis process. After transfected with NRF-2 siRNA, the expression of NRF-2 mRNA and protein were significantly reduced, and the apoptosis rate induced was significantly suppressed （29.90%＋2.45 % vs.43.84%＋14.40%, P=0.001）. Conclusions NRF-2 plays an important role in fenretinide induced apoptosis in NB4 leukemia ceils, suggesting that fenretinide induced apoptosis of NB4 cells through an oxidative stress mediated pathway. By this pathway, elevating the oxidative stress ability of leukemic cells might be a new approach for the targeting therapy of leukemia.