三氧化二砷联合丁硫氨酸亚砜胺对K562/ADM细胞的作用及其机制研究  

The effect of arsenic trioxide(As_2O_3)combined with BSO on K562/ADM cell and its mechanisms

作　　者：王涛[1] 马梁明[1] 张华屏[2] 王宏伟[1] 杨林花[1] 乔振华[1] 

机构地区：[1]山西医科大学第二医院血液科,太原030001 [2]山西医科大学实验中心

出　　处：《中华血液学杂志》2007年第7期438-443,共6页Chinese Journal of Hematology

摘　　要：目的研究三氧化二砷(As_2O_3)联合γ-谷氨酰半胱氨酸合成酶抑制剂丁硫氨酸亚砜胺(BSO)对肿瘤多药耐药细胞株 K562/ADM 细胞的诱导凋亡效应,及对 P 糖蛋白(P-gp)和 mdrlmRNA表达的抑制作用,探讨谷胱甘肽(GSH)的含量变化与 As_2O_3作用效果的关系。方法 As_2O_3(0.5、2.0、5.0 μmol/L)单独及联合100 μmol/L BSO 作用于 K562/ADM 细胞,应用 MTT 比色法检测 K562/ADM细胞的增殖活性;膜联蛋白 V/碘化丙锭(Annexin V/PI)标记法观察 K562/ADM 细胞的凋亡效应;分光光度法检测 K562/ADM 细胞内 GSH 含量变化;流式细胞术(FCM)检测 P-gp 水平变化;RT-PCR 方法检测 mdr1mRNA 的表达变化。结果 K562/ADM 细胞内的 GSH 含量为(81.13±3.91)mg/g 蛋白,在BSO 降低 GSH 含量后,临床剂量(0.5、2.0 μmol/L)As_2O_3联合 BSO(100 μmol/L)24 h内即可抑制K562/ADM 细胞的增殖活性,诱导 K562/ADM 细胞发生凋亡,处理48 h凋亡率分别为(59.29±6.01)%,(65.06±8.29)%;72 h凋亡率分别为(82.15±9.28)%,(92.72±9.41)%;其诱导凋亡效果均明显强于单用 As_2O_3临床剂量和高剂量(5.0 μmol/L)组。K562/ADM 细胞 P-gp 表达阳性率为98.1%,mdr1mRNA 的相对表达水平为1.85±0.13,临床剂量 As_2O_3联合 BSO 处理48 h,其抑制 mdr1mRNA 表达的作用效果以及处理72 h对 P-gp 的抑制作用均明显强于单用高剂量 As_2O_3组。结论GSH 的含量变化与 As_2O_3的作用效果密切相关,As_2O_3联合 BSO 可有效诱导 K562/ADM 细胞发生凋亡,有效抑制 P-gp 及 mdr1mRNA 的表达。Objective To investigate the apoptosis-induction, P-glycoprotein （P-gp） and mdr1 mRNA inhibition effects of arsenic trioxide （As2O3 ） and buthionine sulfoximine （BSO） on muhidrug-resistant cell line K562/ADM cells, and to determine the relationship between intracellular GSH content and arsenic effect. Methods K562/ADM cells were treated with arsenic （ 0.5,2.0,5.0 μmol/L） alone or combined with BSO （ 100μmol/L）. The cell proliferating capacity was assessed with MTT assay, and cell apoptosis by Annexin V and propidium iodide （PI） staining. Intracellular GSH contents were measured using a glutathione assay kit by spectrophotometry. P-gp expression was determined by flow cytometry, and mdrl mRNA expression by semi-quantitative RT-PCR. Results The GSH contents in K562/ADM cell was （81.13 ± 3.91 ） mg/g protein. After the GSH contents were degraded by BSO, the K562/ADM cell proliferating capacity was obviously inhibited and the cells were induced apoptosis in 24 hours by the combination of clinic dose arsenic group（0.5,2.0 μmol/L）and BSO（ 100 μmol/L）. The cell apoptosis rates at 48 hours in arsenic alone group and combination group were （ 59.29 ±6.01 ） % and （ 65.06 ± 8.29 ） % , and at 72 hours were （ 82.15± 9.28） % and（ 92.72 ± 9.41 ） % respectivety. At d.8 hours, the mdrl mRNA inhibition effect of the combination group was obviously stronger than that of high dose arsenic alone group. At 72 hours, the P-gp inhibition effect of the combination group （ clinic dose arsenic group, 0.5,2.0 μmol/L） was obviously stronger than that of high dose arsenic alone group （ 5.0 μmol/L）. Conclusion The intracellular GSH contents are closely correlated with the arsenic effect. The combination of conventional dose arsenic and BSO significantly induces K562/ADM cell apoptosis and inhibits P-gp and mdrl mRNA expression in the cells.

关 键 词：K562/ADM细胞 抗药性 多药 砷剂 谷胱甘肽 细胞凋亡 

分 类 号：R733.7[医药卫生—肿瘤]