过氧化氢诱导人巨核细胞系Dami氧化应激模型的建立及评价  

Hydrogen peroxide- induced oxidative stress model of human megakaryocytic Dami cells and its assessment

作　　者：汪海涛[1] 林洁[1] 杨波[1] 朱宏丽[1] 李素霞[1] 范辉[1] 郭搏[1] 冉海红[1] 翟冰[1] 刘洋[1] 李宝玲[1] 张文英[2] 李建华[3] 杜鑫[3] 

机构地区：[1]解放军总医院南楼血液科,北京100853 [2]解放军总医院生物治疗病区,北京100853 [3]解放军总医院老年心内科,北京100853

出　　处：《解放军医学院学报》2014年第4期349-352,356,共5页Academic Journal of Chinese PLA Medical School

基　　金：科技部"新药创制"重大专项(2008ZXJ09001-019);全军保健专项课题(13BJ247)~~

摘　　要：目的探讨过氧化氢(H2O2)诱导Dami细胞氧化应激模型的条件,建立并评价模型。方法不同浓度过氧化氢处理Dami细胞后,Giemsa染色,显微镜观察细胞形态学的变化;CCK-8试剂盒检测细胞增殖活性,绘制生长曲线,计算半数抑制浓度(IC50);加载DCFH-DA探针后流式细胞仪检测细胞内活性氧自由基水平;Western blot检测核因子NF-E2相关因子(Nrf2)、醌氧化还原酶(NQO1)、血红素加氧酶(HO-1)、γ-谷氨酰半胱氨酸合成酶催化亚单位(GCLC)表达量。结果随着H2O2浓度升高,Dami细胞核皱缩趋于明显,存活率下降,IC50为(0.9132±0.144)mmol/L;0.1 mmol/L、1 mmol/L、10 mmol/L H2O2处理Dami细胞2 h,细胞氧化应激阳性率分别为12.11%、20.91%、52.53%;H2O2处理Dami细胞24 h后,抗氧化蛋白Nrf2、GCLC水平升高,未检测到HO-1和NQO1的表达。结论 1 mmol/L H2O2是建立Dami细胞氧化应激模型的合适浓度。Dami细胞抗氧化应激反应与Nrf2/ARE通路有关。Objective To establish and assess the H2O2 -induced oxidative stress model of Dami cells. Methods Morphology of Giemsa-stained Dami cells was observed under microscope after they were treated with H2O2 at different concentrations. Proliferation of Dami cells was assayed with CCK-8 kit. Growth curve of Dami cells was plotted and 50% inhibiting concentration （IC50） of H2O2 was calculated. ROS levels in Dami cells were measured by Flow cytometry after DCFH-DA probe was loaded. Nrf2, NQO1, HO-1, and γ - GCLC expressions were detected by Western blot. Results The shrinkage of Dami cells increased and their survival rate decreased with the increasing H202 concentration. The IC50 of H202 was 0.9132± 0.144 mmol/L 2 h after the Dami cells were treated at the concentration of 0.1, 1, and 10 mmol/L, respectively. The positive oxidative stress rate of Dami cells was 12.11%, 20.91%, and 52.53%, respectively. The expression levels of Nrf2 and γ-GCLC were higher 24 h after the Dami cells were treated with H2O2. However, no expression of HO-1 and NQOi was detected. Conclusion Oxidative stress model of Dami cells can be established with H2O2 at the concentration of I mmol/L and anti-oxidative stress of Dami cells is associated with the Nrf2/ARE pathway.

关 键 词：过氧化氢 Dami细胞 氧化应激模型 NF—E2相关因子 

分 类 号：R34[医药卫生—基础医学]