三氧化二砷联合阿霉素对人淋巴瘤细胞增殖及凋亡的影响

Effect of arsenic trioxide combined with adriamycin on the proliferation and apoptosis of human lymphoma cells

目的:探讨三氧化二砷(As2O3)联合阿霉素(adriamycin,ADM)对淋巴瘤细胞增殖及凋亡的影响。方法:将Raji细胞分为实验组与对照组,实验组分为1μmol/L As2O3组、2μmol/L As2O3组、ADM组、1μmol/L As2O3与ADM联合组、2μmol/L As2O3与ADM联合组。用As2O3和ADM干预不同时间的人淋巴瘤细胞株Raji,采用Wright-Giemsa染色法观察细胞凋亡的形态学变化;采用噻唑蓝比色法检测As2O3联合ADM对淋巴瘤细胞的增殖抑制作用;流式细胞术检测As2O3联合ADM对淋巴瘤细胞凋亡率及淋巴瘤细胞内ADM荧光强度的影响;半定量聚合酶链反应检测As2O3和ADM对淋巴瘤细胞p53表达的影响。结果:As2O3联合ADM作用细胞24h后,可观察到细胞出现凋亡形态学改变。与As2O3和ADM单药组相比,As2O3联合ADM可增强对Raji细胞的抑制率,差异有统计学意义(P<0.05),其作用呈浓度和时间依赖性;As2O3与ADM联合组与单药组相比,Raji细胞的凋亡率差异具有统计学意义(P<0.05);As2O3和ADM单药组及联合组p53的表达水平均低于对照组,差异有统计学意义(P<0.05);另外,加入1μmol/L和2μmol/L As2O3后细胞内ADM的荧光强度分别为18.53和18.12,分别增加0.056倍和0.023倍,差异无统计学意义(P>0.05)。结论:As2O3和ADM单药及两种药物联合体外均可抑制淋巴瘤细胞增殖,诱导凋亡和下调突变型p53;As2O3和ADM联合体外有协同抗淋巴瘤作用;As2O3对Raji细胞内ADM浓度无显著影响;As2O3可增强淋巴瘤细胞的化疗敏感性,其下调突变型p53的表达可能是其分子水平的作用机制之一。

Objective To determine the effect of arsenic trioxide combined with adriamycin（ADM） on the proliferation and apoptosis of human lymphoma cells.Methods Raji cells were divided into an experimental group and a control group,and the experimental group was further divided into 1 μmol/L As2O3 group,2 μmol/L As2O3 group,ADM group,1 μmol/L As2O3 and ADM group,2 μmol/L As2O3 and ADM group.Human lymphoma cells Raji were treated with As2O3 combined with ADM.Wright-Giemsa dying assay was used to observe the apoptosis morphology of lymphoma cells.The proliferation of the cells treated with As2O3 and adriamycin was detected by the method of 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT）.Flow cytometry（FCM） was used to detect the apoptosis rate of lymphoma and the fluorescene density in the lymphocytes.Effect of arsenic trioxide and adriamycin on the mutant p53 expression in Raji cells was detected by semi-quantitive RT-PCR.Results Evident apoptotic morphological changes of Raji cells were observed 24 hours after treatment with As2O3 or ADM.Compared with As2O3 or ADM alone,As2O3 combined with ADM could increase the inhibition ratio significantly（P〈0.05）,and the inhibition rate was related to the concentration and action time of As2O3（P〈0.05）.Compared with As2O3 or ADM alone,As2O3 combined with ADM could increase the apoptosis rate of lymphoma cells with obvious difference（P〈0.05）.Semi-quantitive and RT-PCR showed that the expression of mutant p53 in As2O3 and ADM alone and combined groups was obviously less than that in the control（P〈0.05）.After the treatment with 1 μmol/L and 2 μmol/L As2O3,the fluorescene density in the Raji cells was 18.53 and 18.12,0.056 and 0.023 times increase respectively.There was no difference（P〉0.05）.Conclusion As2O3 and ADM alone or combined can inhibit the proliferation,induce cell apoptosis,and downregulate the expression of mutant p53 in vitro.As2O3 combined with ADM has synergistic anti-lymphoma cell effect in vitro.As2O3 has no significant effect on the concentration of ADM on the Raji cells,but can enhance the chemosensitivity of Raji cells,and its mechanism may be that it can downregulate the expression of mutant p53.