Disulfiram联合Cu通过上调TNF-α表达及蓄积ROS诱导白血病干细胞凋亡  

Disulfram combined with copper induces leukemia stem cell apoptosis through TNF-α/ROS pathway

作　　者：周勇[1] 钟文彬[2] 陈凯[1] 董慧娟[1] 闫道广[2] 周淑云[1] 徐兵[1] 

机构地区：[1]南方医科大学南方医院血液科,广州510515 [2]暨南大学生命科学技术学院,广州510632

出　　处：《第三军医大学学报》2015年第10期984-989,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81070425);国家自然科学基金青年科学基金(81400104);广东省科技计划项目(2009B050700028)~~

摘　　要：目的探讨Disulfiram联合Cu(DS/Cu)诱导急性髓系白血病(acute myeloblastic leukemia,AML)细胞凋亡及其相关分子机制。方法磁珠筛选CD34+CD38-的KG1α细胞作为AML干细胞,流式细胞术检测空白对照组、DS单药组(5μmol/L)、DS/Cu组(5μmol/L/0.5μmol/L)、DS/Cu+NAC组(5μmol/L/0.5μmol/L/10 mmol/L)的AML干细胞24 h后凋亡比率及ROS水平;qRT-PCR检测上述各组TNF-α、CD40、TNFRSF11B、TNFRSF1B、HRK凋亡相关基因mRNA表达水平;Western blot检测各组处理后TNF-α的蛋白表达水平;予以中和抗体TNF-αm Ab(2μg/m L)和对照抗体Ig G(2μg/m L)预处理2 h后,流式细胞术检测空白对照组、DS/Cu组、TNF-αm Ab组、TNF-αm Ab+DS/Cu组、Ig G组及Ig G+DS/Cu组的AML干细胞24 h后的ROS水平。结果予以DS或DS/Cu处理CD34+CD38-KG1α细胞后,细胞凋亡率由空白对照组的(6.65±0.64)%分别上升至(11.87±1.30)%、(27.43±1.65)%,差异均具有显著性统计学意义(P=0.01和P<0.01);CD34+CD38-KG1α细胞ROS也分别上升了(1.39±0.12)倍和(2.81±0.11)倍,差异也具有统计学意义(P<0.03和P<0.01),并且DS/Cu组比DS组更明显(P=0.00);应用NAC抑制DS/Cu诱导ROS蓄积后,细胞凋亡率由(27.43±1.65)%下降到(12.37±0.85)%(P<0.01)。qRT-PCR结果显示DS及DS/Cu均上调TNF-α、CD40、TNFRSF1B、TNFRSF11B、HRK凋亡相关基因的mRNA水平(P<0.05),且DS/Cu组较DS组上调的更明显(P<0.05),但是予以NAC预处理2 h后,仅TNF-α基因水平依然高表达(P=0.73),而CD40、TNFRSF11B、TNFRSF1B、HRK基因水平均明显下调(P<0.05)。为进一步证实TNF-α作用,分别予以TNF-αm Ab(2μg/m L)或Ig G(2μg/m L)预处理2 h后,TNF-αm Ab+DS/Cu组相比DS/Cu组的ROS相对变化率由(2.78±0.25)倍下降到(1.28±0.17)倍(P=0.00),而Ig G+DS/Cu组相比DS/Cu组的ROS相对变化率无显著性差异(P=0.23),TNF-αm Ab及Ig G组相比空白对照组的ROS水平无显著性差异(P=0.09和P=0.50)。结论 DS/Cu可通过上调TNF-α表达及蓄积ROS诱导AML干细胞凋亡。Objective To investigate the molecular mechanism of disulfram/copper （DS/Cu）- induced reactive oxygen species （ROS） accumulation and apoptosis in leukemia stem cells （LSCs）. Methods CD34 ＋ CD38- KGlα cells were separated from KGlα lines by magnetic cell sorting （MACS）. Then, qRT- PCR was employed for determination of TNF-α, CD40, TNFRSF1 B, TNFRSF11B and HRK mRNA levels, and Western blαting was used to detect TNF-α prαein level. ROS level and cell apoptosis were assayed byflow cytometry. Results The apoptαic proportions of CD34 ＋ CD38- KGlα cells exposed to DS and DS/Cu were （ 11.87 ± 1.30） % and （27.43 ± 1.65） % , respectively （P = 0.01, P 〈 0. 01 ）. DS and DS/Cu could increase ROS accumulation by 1.39 ± 0. 12 folds and 2.81 ± 0. 11 folds, respectively, （P = 0. 03, P 〈 0. 01 ）. However, cell apoptosis induced by DS/Cu was inhibited by pre-treatment of ROS inhibitor N-acetyl- L-cysteine （NAC） [ （27.43 ± 1.65） % vs （ 12.37 ± 0. 85） % , P 〈 0. 01 ]. The mRNA levels of TNF-α , CD40, TNFRSFllB, TNFRSFIB and HRK were up-regulated after treatment with DS and DS/Cu （P 〈 0. 05 ） , and the up-regulation induced by DS/Cu was more significantly （P 〈 0. 05 ）. Pre-treatment of NAC significantly down-regulated the CD40, TNFRSF11B, TNFRSF1 and HRK expression （P 〈 0. 05 ）, but TNF-ct expression was still at high level （P = 0. 726 ）. To further confirm the role of TNF-α, ROS accumulation was inhibited by pre-treatment with neutralizing antibody TNF-α mAb （2.78 ± 0.25 vs 1.28 ± 0. 17 folds, P =0. 00）, while pre-treatment with IgG had no impact on ROS accumulation induced by DS/Cu （P = 0. 23）. Conclusion DS/Cu induces leukemia stem cell apoptosis through TNF-α/ROS pathway.

关 键 词：Disulfram CU CD34+CD38-KG1α TNF-α ROS 

分 类 号：R733.71[医药卫生—肿瘤] R966[医药卫生—临床医学]