雄黄诱导维甲酸耐药的急性早幼粒细胞白血病细胞(MR_2细胞)凋亡的体外研究

In vitro Study on Realgar Induced Apoptosis of All-trans Acid Resistant Acute Promeylocytic Leukemia Cell Line (MR_2)

目的 :深入了解雄黄治疗急性早幼粒细胞白血病 (APL)的机理。方法 :以对全反式维甲酸 (ATRA)耐药的APL细胞株MR2 细胞为体外模型 ,用不同剂量的雄黄处理细胞一定时间后 ,利用MTT实验观察细胞的存活、增殖状态 ,应用细胞形态学、基因组DNA电泳、流式细胞仪检测细胞凋亡 ,利用流式细胞仪分析细胞周期及细胞膜AnnexinⅤ的表达。结果 :一定浓度范围内雄黄对MR2 细胞的生长、增殖有剂量、时间依赖性抑制作用 ;Wright’s及HE染色均可见凋亡细胞 ,透射电镜下可见核染色质边集、核碎裂及凋亡小体 ;基因组DNA电泳出现“梯形”条带 ;流式细胞仪分析出现低于G1期DNA含量的亚二倍体细胞峰 ;经雄黄处理后AnnexinⅤ FITC+ /PI-的凋亡细胞逐渐增多。结论 :雄黄可诱导ATRA耐药的APL细胞发生凋亡 ,提示雄黄治疗APL的效应途径可能不同于ATRA。

Objective: To acquire a deep understanding of the possible mechanisms of realgar in the treatment of acute promyelocytic leukemia (APL). Method: All- trans retinoic acid (ATRA) resistant APL cell line MR 2 was used as in vitro model. The effect of realgar on MR 2 cell was observed by watching cell viability, cell growth, and by using Methy thiazolyl tetrazolium (MTT) assay, cell morphology, DNA gel electrophoresis and flow cytometry assay. Result: The viability and growth of MR 2 cell were inhibited after the treatment, to some extent, in a dose and time dependent manner. After being treated with realgar, MR 2 cell presented morphologically some features of apoptotic cells such as intact cell membrane, chromatin condensation and nuclear fragmentation, and apoptotic body could be found by electron microscopy as well. Sub-G 1 cells were observed by flow cytometry, as well as AnnexinⅤ FITC +/PI -cells. DNA ladder could be found by DNA gel electrophoresis. Conclusion: Realgar can induce apoptosis of ATRA resistant APL cell line MR 2 , Which shows the therapeutic effect of realgar on APL may be different from that of ATRA.