柚皮素对人类白血病K562细胞生长的作用及其机制探讨

 目的 研究柚皮素（naringenin）对人类慢性髓系白血病K562细胞株的作用及其机制。方法 体外培养K562细胞，MTT法检测柚皮素在不同浓度、不同时间点对K562细胞生长的影响；用免疫组织化学的方法检测细胞增殖核抗原（PCNA）的表达，并计算PCNA标记指数（LI）；显微镜和透射电镜下观察细胞形态变化；流式细胞仪分析细胞周期分布和细胞凋亡率；用RT-PCR和Western blot的方法检测K562细胞中p53、p21WAF1 mRNA和蛋白表达的变化。结果 柚皮素对K562细胞增生有明显抑制作用，作用24、48和72 h后的半数抑制浓度（IC50）分别为455 μmol／L、225 μmol／L和175 μmol／L；PCNA在柚皮素实验组的表达显著低于对照组；普通光镜以及电镜观察均见实验组细胞显著的增生抑制和典型的细胞凋亡形态学改变；流式细胞仪分析证实柚皮素能使K562细胞聚积在G0／G1期、S期细胞减少，凋亡率增加；RT-PCR和Western-blot检测表明，实验组细胞表达p21 mRNA和蛋白质呈浓度和时间依赖性升高，而p53变化不明显。结论 柚皮素对K562细胞具有明显的增生抑制作用，细胞周期阻滞和诱导凋亡可能是其重要机制，而非p53依赖性的p21上调可能是其发挥作用的重要途径。

Effect and mechanism of naringenin on the proliferation of human leukemia K562 cell line

Objective To investigate the effect and mechanism of narigenin on human leukemia K562 cell line. Methods K562 cell line cultured in vitro was exposed to different concentration of narigenin and cell proliferation manner was detected by MTT assay, and cell PCNA protein was detected by immunhis－tochemical staining. K562 cell morphology and apoptosis were observed by light microscope and electron mi－croscope, and flow cytometry was used to decide cell cycle and apoptosis rate. Expression of p53 and p21WAF1 mRNA and protein was detected by RT-PCR and Western blot technology respectively. Results Proliferation of K562 cell was significantly inhibited by nerigenin and the IC50 for 24 h, 48 h and 72 h were 455 μmol/L, 225 μmol/L and 175 μmol/L respectively. PCNA protein in treated groups was remarkably poorer than in con－trol group, cell growth-inhibition and morphologic change of apoptosis were observed under light microscope and electron microscope. Observation by flow cytometry indicates that K562 cells were blocked in G0/G1 phase of cell cycle in nerigenin group and the apoptosis rates were remarkably increased. Both RT-PCR and West－ern blot tests showed gradually higher expression of p21/WAF1 gene in treated group, whereas p53 gene ex－pression no detectable change between control group and nerigenin group. Conclusion Nerigenin has signifi－cant inhibitory effect on the growth of K562 cell in vitro; Cell cycle blockage and apoptosis induce maybe the important mechanisms underlied; and a p53-independent up regulation of p21/WAF1 may be one of an im－portant pathway during the process.