高三尖杉酯碱联合三氧化二砷诱导人多发性骨髓瘤RPMI 8226细胞株凋亡的实验研究

目的通过体外实验阐述高三尖杉酯碱（HHT）单药及联合亚砷酸（ATO）用药对人多发性骨髓瘤细胞株RPMI 8226作用及其机制。方法应用四甲基偶氮唑（MTT）比色法，检测HHT、ATO单药及两者联合用药对人多发性骨髓瘤细胞株RPMI 8226增殖的影响，Hoechst染色法检测细胞凋亡形态学变化，流式细胞仪检测细胞早期凋亡比率。Western blot 检测药物处理后凋亡关键蛋白（Caspase-3、9及PARP）、Bcl-2家族蛋白（Bcl-2、Mcl-1、Bcl-xl）及AKT蛋白的表达。结果HHT、ATO单药可显著抑制RPMI 8226细胞的增殖，其作用呈时间及浓度依赖（P<0.05），且两药联合具有协同作用（CI<1）。HHT、ATO单药可诱导RPMI 8226细胞凋亡且呈浓度依赖，出现细胞凋亡形态学改变及早期凋亡比率增高，两药联合可加强诱导凋亡作用。HHT可呈浓度依赖性激活Caspase-3、PARP表达；HHT（40 ng/mL）与ATO（8.5 μmol/L）联合用药可显著激活Caspase-3、9，下调抗凋亡蛋白Mcl-1及Bcl-xl的表达，呈时间依赖性降低AKT磷酸化水平。结论HHT、ATO单药或联合用药可诱导RPMI 8226细胞凋亡，其机制可能和激活Caspase通路、调节Bcl-2家族蛋白表达、抑制AKT磷酸化等有关。

英文摘要:

ObjectiveTo clarify the effects and mechanisms of homoharringtonine （HHT） monomer therapy or combination therapy with arsenic trioxide （ATO） on human multiple myeloma （MM） cell line RPMI 8226 in in vitro researches. MethodsEffects of HHT， ATO， and HHT combined ATO on the growth of MM cell line RPMI 8226 were detected using MTT assay. The morphological changes of cell apoptosis were detected by Hoechst staining. The early apoptosis rate was detected using flow cytometry. Expressions of Caspase-3， Caspase-9， poly-ADP-ribose polymerase （PARP）， Bcl-2， Mcl-1， Bcl-xl， and AKT protein were detected by Western blot. ResultsHHT and ATO inhibited the proliferation of RPMI 8226 cell line in a time-and dose-dependent manner （P<0.05）. Synergistic effects was shown in the combination group （CI<1）. HHT and ATO induced the apoptosis of RPMI 8226 in a dose-dependent manner with typical morphological changes of apoptosis and higher early stage apoptosis rate. The enhancement in apoptotic induction was seen when two agents were combined. HHT activated expressions of Caspase-3 and PARP in a dose dependent manner at 24 h. HHT at 40 ng/mL and ATO at 8.5 μmol/L could significantly activate expressions of Caspase-3 and Caspase-9， and down-regulate expressions of anti-apoptotic proteins Bcl-xl and Mcl-1. In addition， the combination therapy of HHT at 40 ng/mL and ATO at 8.5 μmol/L inhibited phosphorylation of AKT in a time-dependent manner. ConclusionHTT， ATO， and combination therapy of HHT and ATO induced the apoptosis of RPMI 8226 cell line possibly through activating Caspase pathways， regulating expressions of Bcl-2 families， and inhibiting phosphorylation of AKT.

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