单甲基亚砷酸联合隐丹参酮抑制多发性骨髓瘤U266细胞增殖作用机制的研究  

Mechanism of Inhibiting Proliferation of Multiple Myeloma U266 Cells by Monomethylarsenous Acid Combined with Cryptotanshinone

作　　者：李爽 邹建华 叶晓鸥 张光霁[2] 陈喆[3] LI Shuang;ZOU Jianhua;YE Xiaoou;ZHANG Guangji;CHEN Zhe(First Clinical Medical College,Zhejiang University of Traditional Chinese Medicine,Hangzhou,Zhejiang province,310051,China;Department of TCM Internal Medicine,Zhejiang Hospital of Traditional Chinese Medicine,Hangzhou,Zhejiang province,310002,China;Central Laboratory,Zhejiang Hospital of Traditional Chinese Medicine,Hangzhou,Zhejiang province,310002,China.)

机构地区：[1]浙江中医药大学第一临床医学院,杭州310051 [2]浙江省中医院中医内科,杭州310002 [3]浙江省中医院中心实验室,杭州310002

出　　处：《浙江中西医结合杂志》2019年第3期181-185,I0004,I0005,共7页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：国家自然科学基金资助项目(No.81473389)

摘　　要：目的研究单甲基亚砷酸(MMAIII)联合隐丹参酮(CPT)对多发性骨髓瘤U266细胞的协同抑制效应,阐明其诱导多发性骨髓瘤凋亡的作用机制。方法多发性骨髓瘤U266细胞用含10%gibco胎牛血清的RPMI1640培养液培养,按隐丹参酮单药组、MMAIII单药组和联合组分别加药处理。cell-counting-kit-8(CCK8)法检测细胞存活率;AnnexinⅤ-FITC/PI流式细胞术检测细胞凋亡;Western Blot检测凋亡标记蛋白PARP的总蛋白和剪切表达水平;酶标仪检测酸性鞘磷脂酶活性;免疫荧光技术检测细胞膜神经酰胺和脂筏标记物。结果 CCK8法检测结果显示,隐丹参酮浓度15μM时细胞存活率为(80.9±5.4)%(24h)、(60.0±8.4)%(48h),单甲基亚砷酸浓度1μM时细胞存活率为(82.5±5.8)%(24h)、(67.7%+9.7)%(48h),隐丹参酮(15μM)联合单甲基亚砷酸(1μM)作用U266细胞24h、48h后细胞存活率为(29.1±7.0)%(24h)、(18.0±2.7)%(48h);流式细胞术结果显示,联合用药组细胞凋亡率为(41.7±4.4)%、单甲基亚砷酸组为(10.6±4.0)%、隐丹参酮组为(10.5±3.0)%;Western Blot显示,联合作用组凋亡蛋白PARP剪切活化水平较单甲基亚砷酸单药组及隐丹参酮单药组有显著升高;酸性鞘磷脂酶活性测定结果显示,联合用药组上调多发性骨髓瘤U266细胞酸性鞘磷脂的活性;免疫荧光技术检测结果显示,联合用药后多发性骨髓瘤U266细胞膜上荧光强度增加,加入Filipin后荧光强度较联合用药减少。结论 MMAIII与CPT协同作用能诱导人多发性骨髓瘤U266细胞凋亡发生,其作用机制主要与促进细胞膜鞘磷脂水解生成神经酰胺、诱导脂筏聚集促进凋亡相关蛋白剪切活化密切相关。Objective To explore the mechanism of synergistic inhibition effect on multiple myeloma U266 cells and the induction of apoptosis in multiple myeloma by monomethylarsenous acid(MMAIII)combined with cryptotanshinone(CPT).Methods Multiple myeloma U266 cells were cultured with RPMI1640 medium containing 10%GIBCO fetal bovine serum,and treated with CPT,MMAIII,and CPT+MMAIII,respectively.The cell viability was assessed by cell-counting-kit-8(CCK8).Annexin V-FITC/PI assay was used to detect the apoptosis of cells U266.Western blot was used to evaluate the total protein and splicing expression of apoptosis-related protein PARP.Acid sphingomyelinase activity was detected by enzyme labeling instrument.Cell membrane ceramide and lipid raft markers were tested by immunofluorescence technique.Results The CCK8 assay showed that the cell survival rate was(80.9±5.4)%(24h)and(60.0±8.4)%(48h)when treating with 15μM CPT,and was(82.5±5.8)%(24h)and(67.7+9.7)%(48h)when treating with 1μM MMAIII,and was(29.1±7.0)%(24h)and(18.0%±2.7)%(48h)when treating with 15μM CPT combined 1μM MMAIII.The Flow cytometry assay revealed that the apoptotic rate were(41.7±4.4)%in the combination group,(10.6±4.0)%in MMAIII group,and(10.5±3.0)%in CPT group.Western blotting showed that the level of PARP cleavage activation in the combination group increased significantly,compared to those of MMAIII group and CPT group.The acid sphingomyelinase activity was up-regulated in the combination group.The fluorescence intensity of U266 cells increased after treating with the combination of MMAIII and CPT,and its fluorescence intensity decreased after adding Filipin.Conclusion The combined treatment of MMAIII and CPT could co-induce the apoptosis of human multiple myeloma U266 cells,its mechanism mainly related to the promotion of ceramide production and the aggregation lipid raft to enhance the apoptosis-related protein shear activation.

关 键 词：单甲基亚砷酸 隐丹参酮 多发性骨髓瘤U266细胞 神经酰胺 脂筏 

分 类 号：R733.3[医药卫生—肿瘤]