雄黄微生物提取液诱导K562/ADM细胞凋亡的作用机制研究

Realgar Bioleaching Solution Induced Apoptosis in K562/ADM Cells and its Mechanisms

目的:观察雄黄微生物提取液对K562/ADM细胞的诱导凋亡作用,探讨其作用机制、并初步探寻雄黄溶解后表现药理活性的化学物种。方法:应用噻唑蓝(MTT)比色法、Annexin V-PI双染法染色、DNA琼脂糖凝胶电泳和流式细胞术(FCM)观察K562/ADM细胞凋亡,FCM测定K562/ADM细胞Fas、Bcl-2、Caspase3的活性变化,并以H3AsO3(As2O3在该溶液中的主要存在形式)作为阳性对照。结果:雄黄微生物提取液可抑制K562/ADM细胞增殖;K562/ADM呈典型凋亡形态改变;DNA电泳可见梯状条带出现;FCM分析显示:G1期细胞比例增高,G2-M和S期阻滞;Fas蛋白表达明显上调、Bcl-2蛋白表达显著下调、Caspase-3活性明显增强。结论:雄黄微生物提取液可通过Fas/Bcl-2途径,激活Caspase-3而诱导K562/ADM细胞凋亡。砷酸(V)、甲基砷酸盐(V)可能是雄黄溶解后表现药理活性的主要化学物种。

Objective：To observe the apoptosis of K562/ADM cells induced by realgar bioleaching solution and to explore its possible mechanisms ,and to find out the chemical species with efficacy. Methods：MTT assay,morphological observation with Annexin V-PI double staining,DNA agarose gelelectrophoresis and cell cycle analysis were used to examine apoptosis in K562/ADM cells. Expression levels of Fas,Bcl-2 and Caspase -3 protein were measured with FCM. Results：RBS inhibited growth of K562/ADM cells. Morphological changes typical of apoptosis were observed through Laser Scanning Confocal Microscopy. Agarose gelelectrophoresis showed evident DNA fragmentation. Cell cycle analysis indicated increased Sub-G1 proportion and apoptosis rate,as well as apparent G2-M and S phase arrest. The expression of Fas protein significantly increased and Bcl-2 protein largely decreased after application of RBS. Caspase-3 was also activated by RBS. Conclusion：RBS induces apoptosis in K562/ADM cells by activating Caspase-3 via a Fas/Bcl-2 dependent pathway. Both H3ASO3 and MMAV may be the active intermediates in the dissolved solution of realgar.