葛根总黄酮诱导白血病细胞株凋亡及其分子机制的研究

 目的　探讨葛根总黄酮（PR）对慢性粒细胞白血病（CML）细胞株K562和急性早幼粒细胞白血病（APL）细胞株NB4细胞增殖及凋亡的影响。方法　采用MTT法检测PR对K562细胞、NB4细胞的增殖抑制率；光学显微镜及荧光显微镜观察细胞形态改变；Hoechest33258荧光染色AnnexinV／PI双染法检测细胞凋亡率；DNA PI染色法分析细胞周期及亚二倍体峰。Western blot分别检测NB4细胞JNK、PARP、bcl-2、Caspase3，K562细胞bcr-abl、p53、bcl-2、Fas／FasL蛋白表达的变化。结果　12.5～200 μg／ml PR均能抑制K562、NB4细胞增殖。光学显微镜及荧光显微镜下观察到核固缩、凋亡小体等典型的细胞凋亡改变；Annexin V+／PI-细胞呈时间-剂量依赖性增加；DNA PI染色法发现细胞亚二倍体比例增加，G1期比例下降、S期比例增加。PR呈时间-剂量依赖性抑制K562细胞、NB4细胞增殖，诱导细胞凋亡。不同浓度PR干预后K562细胞bcr-abl蛋白水平呈浓度依赖性下调（F＝18.74，P＜0.05），而bcl-2则无明显变化；p53 表达呈浓度依赖性上调；Fas／FasL 表达无明显变化。NB4细胞JNK、PARP及Caspase 3蛋白表达与PR浓度呈正相关，与凋亡抑制蛋白bcl-2则呈负相关（F＝42.32，P＜0.05）。结论　PR能有效抑制K562、NB4细胞增殖，阻滞细胞周期进程，诱导细胞凋亡，但分子机制不同。提示一定浓度PR具有较广谱的抗白血病效应。

Apoptosis of leukemic cells induced by flavonoids of puerarin and its molecular mechanisms

Objective To explore the effects and the possible molecular mechanism of flavonoids of puerarin （PR） on chronic myelogenous leukemia （CML） cell line K562 and acute promyelocytic leukemia （APL） cell line NB4 in vitro. Methods MTF assays were used to detect the inhibitory effects of cell proliferation. The apoptosis of K562 and NB4 ceils was detected by flow cytometry marked with AnnexinV/PL The expression of bcr-abl, p53, bcl-2, Fas/FasL in K562 cells and JNK, PARP, bcl-2 and Caspase 3 in NB4 cells at protein level was detected by Western blot. Results PR could inhibit the proliferation of K562 and NB4 cells in a time-dose dependent manner. The expression of protein levels of bcr-abl fusion gene declined, while the p53 protein otherwise increased, and both were in a dose-dependent manner （F =18.74, P 〈0.05）. The application of PR had no effect on bcl-2 and Fas/FasL protein expression in K562 cells. The JNK, PARP and Caspase3 proteins were upregulated in NB4 cells, while bcl-2 was downregulated with the increasing concentrations of PR （F=42.32, P 〈0.05）. Conclusion PR could inhibit leukemic cell proliferation, induce cell cycle block, and increase cell apoptosis through different molecular mechanisms. It suggestes that PR might potentially be a kind of broad spectrum anti-leukemia agent.