雄黄诱导人弥漫性大B淋巴瘤SU-DHL-4细胞凋亡及其机制的初步研究

本研究旨在探讨雄黄（Realgar，AS4S4）诱导人弥漫性大B淋巴瘤su-DHL-4细胞凋亡及其可能的机制。采用MTT法观测雄黄对SU—DHL-4细胞的增殖抑制作用，应用流式细胞术检测药物对SU-DHL-4细胞周期的影响及其诱导凋亡情况，利用琼脂糖凝胶电泳方法观察凋亡细胞的DNA降解情况，通过透射电子显微镜技术观察药物作用前后细胞超微结构的变化，采用Westernblot方法检测药物诱导SU—DHL4细胞48h后Caspase-3、Bcl-2以及Bax蛋白表达情况。结果表明：20、40、80μmol／L的雄黄均可抑制su—DHL-4细胞增殖，抑制率呈时间-剂量依赖性（r=0．982；P〈0．05）；AnnexinV／PI双染流式细胞术检测细胞凋亡率显示雄黄作用SU—DHL-4细胞48h后，随着药物浓度的增加，早期凋亡细胞所占比率增高（P〈0．05）；PI单染流式细胞术检测细胞周期发现，雄黄作用SU—DHL4细胞48h后处于细胞周期S期的细胞显著减少（P〈0．05）；透射电子显微镜下观察雄黄作用SU-DHL-4细胞48h后各浓度组细胞，均呈典型凋亡的改变，偶见坏死细胞；琼脂糖凝胶电泳结果显示，各浓度实验组雄黄干预SU—DHL-4细胞48h后，细胞核DNA降解呈梯状；Westemblot结果显示，雄黄作用SU-DHL-4细胞48h后，Bcl-2蛋白表达降低，Bax和Caspase-3蛋白表达增加。结论：雄黄可以诱导人弥漫性大B淋巴瘤细胞凋亡，其作用机制可能是通过抑制细胞增殖、阻滞细胞周期、上调Caspase-3和Bax蛋白，下调Bcl-2蛋白而诱导细胞凋亡。

Effect of Realgar on Induction of Apoptosis in DLBCL Cell Line SU-DHL-4 and Its Possible Mechanisms

This study was aimed to explore the effect of realgar （ As4S4 ） on growth inhibition and apoptosis induction of DLBCL cell line SU-DHL-4 and its mechanisms. The inhibitory effect of realgar on the cell growth were detected by MTT method. The morphological changes of SU-DHL-4 were observed by transmission electron microscopy（TEM）. The apoptosis of SU-DHL-4 cells treated with realgar were detected by flow cytometry with Annexin V-FITC/PI double staining and DNA agarose gelelectrophoresis. The cell cycle was examined by flow cytometry with PI staining. The expressions of apoptosis-related proteins（ BCL-2 , Caspase-3 ,BAX） were detected by Western blot. The results showed that the realgar at the concentration of 20,40,80 μmol/L all could inhibit the proliferation of SU-DHL-4 （P 〈 0.05 ）, and in a certain time and concentration range, the inhibition rate was enhanced in a time and dose dependent manner （ r = 0. 982）. Flow cytometric test results showed that realgar could induce SU-DHL-4 cell apoptosis after treating for 48 hours, and the apoptosis rate increased with the increasing of drug concentration （P 〈 0.05）. After treating SU-DHL-4 cells with Reaigar for 48 h, the cell cycle was blocked in the S phase（P 〈0.05 ）. TEM results revealed that when treated with realgar for 48 h, the typically apoptosis morphology-apoptotic bodies were observed in all drug-treated group, furthermore, some necrotic cells in the 80 μmol/L group were observed. After intervened by realgar for 48 h, the DNA Ladder pattern was seen according to agarose gelelectrophoresis. Western blot showed that the expression of Bcl-2 protein was down-regulated while the expressions of BAX and Caspase-3 protein were up-regulated when treating SU-DHL-4 cells with realgar for 48 h.It is concluded that realgar can inhibit cell growth and induce cell apoptosis, which may be related with up-regulation of Caspase-3 and BAX expression and down-regulation the of BCL-2 expression.