| 李爽,邹建华,叶晓鸥,陈喆,张光霁.单甲基亚砷酸联合隐丹参酮抑制多发性骨髓瘤U266增殖的作用机制研究[J].浙江中西医结合杂志,2019,29(3): |
| 单甲基亚砷酸联合隐丹参酮抑制多发性骨髓瘤U266增殖的作用机制研究 |
| Mechanism of monomethylarsenous acid combined with cryptotanshinone inhibiting the proliferation of multiple myeloma U266 |
| 投稿时间：2018-10-04 修订日期：2018-12-29 |
| DOI： |
| 中文关键词: 单甲基亚砷酸 ,隐丹参酮,多发性骨髓瘤,神经酰胺,脂筏 |
| 英文关键词:monomethylarsenous acid,cryptotanshinone,multiple myeloma, ceramide,lipid raft |
| 基金项目:国家自然科学基金项目（面上项目，重点项目，重大项目） |
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| 中文摘要: |
| 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：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结果显示隐丹参酮浓度为15μM时细胞存活率为80.9%±5.4%(24h)、60%±8.4%(48h)，单甲基亚砷酸浓度为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%±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。 |
| 英文摘要: |
| ABSTRACT Objective：We aimed to illustrate the mechanism that monomethylarsenous acid(MMAIII) combined with cryptotanshinone(CPT) induces apoptosis of multiple myeloma cells.Method：The cell viability was assessed by CCK8.Annexin V-FITC/PI assay was used to detect the apoptosis of human multiple myeloma cells U266.Western blot was used to evaluate the activation of apoptosis-related protein PARP in U266 cells.Ceramide accumulation on cell membrane was assessed by immunofluorescence detection.Microplate reader detects the activity of acid sphingomyelinase.Immunofluorescence technique was used to V detect ceramide colocalization with lipid raf.Results：The CCK8 results showed that the cell survival rate was 80.9%±5.4%(24h) and 60%±8.4%(48h) when the concentration of cryptotanshinone was 15μM,and the cell survival rate was 82.5%±5.8%(24h) and 67.7%+9.7%(48h)when the concentration of monomethylarsenite was 1μM.Cryptotanshinone(15μM) combined with monomethylarsenite(1μM) for 24h and 48h,cell viability was 29.1%±7.0%(24h) and 18% ±2.7%(48h).Flow cytometry showed that the apoptotic rate was 41.7%±4.4% in the combination group,10.6%±4.0% in the monomethylarsenite group,and 10.5%±3.0% in the cryptotanshinone group.Western Blot showed that the level of PARP cleavage activation of the combination group was significantly higher than that of the monomethylarsenite monotherapy group and the cryptotanshinone monotherapy group.The acid sphingomyelinase activity assay showed that the combination group up-regulated the activity of acid sphingomyelin in U266 cells.The results of immunofluorescence assay showed that the fluorescence intensity of U266 cells increased after used the combine of monomethylarsenite and cryptotanshinone ,and the fluorescence intensity decreased after the addition of Filipin.Conclusion：The combined use of MMAIII and CPT induce apoptosis of human multiple myeloma U266 cells，its mechanism of action is mainly related to the promotion of ceramide production and lipid raft aggregation to induce apoptosis-related protein shear activation. |
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