二硫化二砷联合伊马替尼诱导慢性髓细胞白血病细胞凋亡机制探讨

目的：观察二硫化二砷（As2S2）和伊马替尼（STI571）联合用药对慢性髓细胞白血病（chronic myeloid leukemia,CML）细胞株的作用机制。方法：应用MTT增殖抑制实验、锥虫蓝拒染细胞计数、瑞氏染色细胞形态观察、Annexin V流式细胞仪检测凋亡细胞,RT-PCR检测bcr-abl mRNA的表达及蛋白印迹（Western blot）分析BCR-ABL的表达、细胞色素C（cytoC）和天冬氨酸特异性半胱氨酸蛋白酶（caspase）蛋白表达,观察As2S2、STI571联合或单独作用于STI571敏感细胞株K562S及耐药细胞株K562R的情况。结果：2.5μmol/LAs2S2＋0.25μmol/LSTI571或4μmol/LAs2S2＋8μmol/LSTI571联合用药对K562S或K562R有明显生长抑制协同作用和促凋亡协同作用;联合用药对K562S或K562R的bcr-abl mRNA的表达无明显影响,但K562S细胞BCR-ABL表达明显减少,K562R细胞BCR-ABL表达略有减少。且联合用药可促进凋亡诱导蛋白cytoC释放增多及caspase9和caspase3前体下调。结论：STI571与As2S2联合用药对K562S和K562R细胞有明显的生长抑制和促凋亡的协同作用,这一作用可能通过减少BCR-ABL表达及凋亡相关蛋白cytoC释放,并下调caspase9和caspase3前体的表达以促进细胞凋亡。

Effect of As2S2 combination with imatinib on the apoptosis of chrome myeloid leukemic cells

Objective To observe the effect of As2S2 combined with imatimib （STI571） on the apoptosis of chronic myeloid leukemic （CML）cells. Methods Cell apoptosis was assessed by MTT proliferation inhibition test, trypan blue exclusion cell count, Giemsa stain cell morphology and Annexin V flow cytometry, expression of bcr-abl mRNA was determined by RT-PCR, and expression of BCR-ABL was assessed by Western blot.; cytochrome C （cyto C） and caspase were also assessed. The effect of As2S2, STI571 and As2S2 combined with STI571 on STI571 sensitive cell line K562S and STI 571 resistant cell line K562R was appraised by the abovementioned parameters. Results As2S2 2.5 μmol/L plus STI571 0.25 μmol/L or As2S2 4 μmol/L plus STI571 8 μmol/L had obvious synergistic growth inhibiting effect and apoptosis promoting effect on both the K562S and K562R cell lines. Combined use of As2S2 and STI571 had no significant effect on the expression of bcr-abl mRNA, however, the BCR-ABL expression decreased significantly in K562S cells and slightly in K562R cells. Furthermore, the combined use of As2S2 and STI571 enhanced the release of cyto C and downregulated the precursors of caspase 9 and caspase 3. Conclusions Combined use of As2S2 and STI571 had significant synergistic growth inhibiting effect and apoptosis promoting effect on K562S and K562R cells. This effect may be mainly through the decrease of BCR-ABL, the release of cyto C and the downregulation of expression of precursors of caspase 9 and caspase 3, promoting the apoptosis of CML cells.