三氧化二砷诱导多发性骨髓瘤细胞U266自噬及凋亡作用研究

Effects of arsenic trioxide on autophagy and apoptosis of multiple myeloma cell line U266

目的探讨三氧化二砷(As2O3)诱导多发性骨髓瘤细胞U266自噬及凋亡的作用。方法选取多发性骨髓瘤细胞株U266细胞,随机分为对照组、0.3μg/ml组、0.6μg/ml组和1.2μg/ml组,分别为0.9%氯人钠溶液和0.3μg/ml、0.6μg/ml、1.2μg/ml As2O3,采用MTT法检测各组细胞增殖,流式细胞仪检测各组细胞凋亡,透射电镜观察各组细胞自噬,Western blot检测Beclin-1、Caspase-3、Bcl-2蛋白表达。结果随As2O3浓度增加,U266细胞增殖抑制情况越严重,1.2μg/ml组培养24 h、48 h和72 h时OD值分别为(0.382±0.097)、(0.534±0.111)和(0.604±0.121),明显低于0.3μg/ml组、0.6μg/ml组和对照组(P<0.05);0.3μg/ml组、0.6μg/ml组、1.2μg/ml组细胞凋亡率高于对照组(P<0.05),其中1.2μg/ml组细胞凋亡率为(25.43±3.88)%,明显高于0.3μg/ml组、0.6μg/ml组(P<0.05);1.2μg/ml组可见较多特征性的自噬小体;0.3μg/ml组、0.6μg/ml组、1.2μg/ml组Beclin-1相对表达量高于对照组(P<0.05),而Caspase-3、Bcl-2相对表达量低于对照组(P<0.05),其中1.2μg/ml组Beclin-1相对表达量高于0.3μg/ml组、0.6μg/ml组(P<0.05),而Caspase-3、Bcl-2相对表达量低于0.3μg/ml组、0.6μg/ml组(P<0.05)。结论As2O3诱导多发性骨髓瘤细胞U266自噬及凋亡,存在剂量依赖关系,可能与调控Beclin-1、Caspase-3、Bcl-2蛋白表达有关。

Objective To investigate the effects of arsenic trioxide(As2O3)on autophagy and apoptosis of multiple myeloma cell line U266.Methods The U266 cells were randomly divided into control group,0.3g/ml group,0.6g/ml group and 1.2g/ml group,which were given normal saline and 0.3g/ml,0.6g/ml and 1.2g/ml As2O3,respectively.MTT assay was used to detect the cell proliferation,and flow cytometry was used to detect cell apoptosis,and transmission electron microscopy was used to observe cell autophagy,Western Blot was used to detect the expression levels of Beclin-1,Caspase-3 and Bcl-2 protein.Results With the increase of As2O3 concentration,the inhibition of U266 cell proliferation became more serious,and the OD values at 24h,48h and 72h in 1.2g/ml group were(0.382±0.097),(0.534±0.111)and(0.604±0.121),respectively,which were significantly lower than those in 0.3 g/ml group,0.6g/ml group and control group(P<0.05).The cell apoptotic rate in 0.3g/ml group,0.6g/ml group and 1.2g/ml group was significantly higher than that in control group(P<0.05).In 1.2g/ml group,more characteristic autophagic bodies were observed.The relative expression levels of Beclin-1 in 0.3g/ml,0.6g/ml and 1.2 g/ml groups were significantly higher than those in control group(P<0.05),however,the relative expression levels of Caspase-3 and Bcl-2 were significantly lower than those in control group(P<0.05),moreover,the relative expression levels of Beclin-1 in 1.2g/ml group were significantly higher than those in 0.3g/ml group and 0.6g/ml group(P<0.05),but the relative expression levels of Caspase-3 and Bcl-2 were significantly lower than those in 0.3g/ml group and 0.6g/ml group(P<0.05).Conclusion As2O3 can induce the autophagy and apoptosis of multiple myeloma cells U266,with a dose-dependent relationship,which may be related to its regulatory effects on the expression levels of Beclin-1,Caspase-3 and Bcl-2 protein.