新型免疫抑制剂FTY720诱导K562细胞凋亡及其机制

目的：探讨新型免疫抑制剂FTY720诱导K562细胞凋亡的效应及其机制，为临床白血病的疗提供实验依据。方法：体外培养人白血病K562细胞，分为空白对照组和FTY720处理组，FTY720处理组细胞分别采用6 μmol?L^-1 FTY720诱导3、6和12 h，或采用2、4、6和8 μmol?L^-1 FTY720处理24 h。采用流式细胞术分析细胞凋亡百分率、活性氧（ROS）水平、线粒体膜电位（MMP）和细胞周期。采用WST-1 还原法检测FTY720作用下K562细胞增殖抑制率。结果：流式细胞术检测，与空白对照组比较，6 μmol?L-1 FTY720诱导细胞3、6和12 h后，细胞凋亡率随时间延长而升高（P<0.01）；与空白对照组比较，2、4、6和8 μmol?L^-1 FTY720诱导细胞24 h 后，细胞凋亡率随药物浓度增加而升高（P<0.01）。与空白对照组比较，随FTY720浓度增加，K562细胞内ROS水平增加（P<0.01），同时其MMP下降（P<0.01）。细胞周期分析，与空白对照组比较，随FTY720浓度增加，G0/G1期细胞百分率增加（P<0.05），而S期和G2/M期细胞百分率减少（P<0.05）。WST-1 还原法分析，与空白对照组比较，FTY720处理72 h后，2、4、6和8 μmol?L-1FTY720处理组K562细胞增殖抑制率［（24.0±4.1）%、(46.4±3.9)%、(67.0±4.8)%和（88.2±5.6）%］明显升高（P<0.01），FTY720对K562细胞的半数抑制浓度（IC50）为5.5　μmol?L^-1。结论：FTY720可通过导致细胞周期阻滞和激发ROS而诱导细胞凋亡来发挥抗肿瘤作用。

Apoptosis of K562 cells induced by a new immunosuppressive agent FTY720 and its mechanism

Objective To study the apoptosis of K562 cells induced by a new immunosuppressive agent FTY720 and its mechanism,and to provide experimental basis for the treatment of leukemia in clinic.Methods The K562 cells were cultured in vitro and divided into blank control group and FTY720 treatment group.The K562 cells in FTY720 treatment group were treated with 6μmol·L-1 FTY720 for 3,6,and 12 h,or treated with different concentrations of FTY720(2,4,6,8μmol·L-1)for 24 h.The apoptosis,level of reactive oxygen species(ROS),mitochondrial membrane potential(MMP)and cell cycle were measured by flow cytometry.The inhibitory rate of proliferation of K562 cells after treated with FTY720 was detected by WST-1reducting assay.Results The results of flow cytometry showed that the percentages of apoptotic cells were increased after treated with6μmol·L-1 FTY720 for 3,6,and 12 hwith the prolongation of time compared with blank control group(P<0.01).The percentages of apoptotic cells were also increased after treated with different concentrations of FTY720 for 24hcompared with blank control group(P<0.01).Compared with blank control group,the ROS levels were increased with the increasing of FTY720 concentration,while the MMP was decreased(P<0.01).Compared with blank control group,the percentage of cells at G0/G1 phase was increased,while those at S and G2/M phases were decreased with the increasing of FTY720concentration(P<0.05).The WST-1reduction assay results indicated that the inhibitory rates of proliferation of K562 cells after treated with 2,4,6,and 8μmol·L-1 FTY720 for 72h were(24.0±4.1)%,(46.4±3.9)%,(67.0±4.8)%,and(88.2±5.6)%,respectively,compared with blank control group.The concentration of FTY720 to result the inhibitory rate of 50%(IC50)on K562 cells was5.5μmol·L-1.Conclusion FTY720 could inhibit the proliferation of K562 cells by blocking cell cycle and inducing apoptosis through provoking ROS.