大黄素联合AZT对白血病KG-1a细胞的增殖及BCL-2、NF-κB、TGF-β表达的影响

Effect of Emodin Combined with AZT on the Proliferation and the Expression of BCL-2、NF-κB、TGF-β in the Leukemia Stem Cells——KG-1a cells

目的:探讨中药大黄素(Emodin)和端粒酶抑制剂AZT(3'-azido-3'-deoxythymidine)联合应用对浓集白血病干细胞(concentrated leukemia stem cells,CLSC)的急性髓系白血病KG-1a细胞增殖及BCL-2、NF-κB、TGF-β表达的影响。方法:应用5-FU富集白血病KG-1a细胞中肿瘤样干细胞亚群并采用流式细胞术检测富集后CD34+CD38-亚群细胞;采用MTT法检测大黄素和AZT单用及联合应用对CLSC的生长增殖的影响;采用流式细胞术检测药物诱导CLSC的凋亡情况;采用RT-PCR及Western blot法分别检测NF-κB、BCL-2、TGF-βmRNA和蛋白表达变化。结果:大黄素和AZT联合后,细胞生长抑制明显大于大黄素和AZT单药组(P<0.01),具有一定的时间和浓度依赖性;细胞凋亡比例均较各单药组增多,差异具有统计学意义(P<0.01);NF-κB、BCL-2、TGF-βmRNA及蛋白表达在联合组比在各单药组表达量下降。结论:大黄素联合AZT具有协同抑制CLSC增殖并诱导其凋亡的作用,其机制可能与抑制BCL-2活化及降低NF-κB、TGF-β表达有关。

Objective: To investigate the effect of Emodin combined with 3’-azido-3’-deoxythymidine( AZT) on the proliferation and apoptosis of concentrated leukemia stem cells( CLSC) ——human acute myeloid leukemia KG-la cells and expression of BCL-2,NF-κB and TGF-β. Methods: The tumor stem cell-like subpopulation in human leukemia cell line KG-1a was enriched with 5-fluorouracil( 5-FU). The CD34 + CD38- subpopulation in the KG-1a cells was detected with flow cytometry,the cell proliferation was detected by MTT method to study the of Emodin and AZT in the CLSC.The cell apoptosis was analyzed by flow cytometry. The expression of NF-κB,BCL-2 and TGF-β mRNA and proteins were measured with RT-PCR and Western blot respectively. Results: As compared with cells treated with mentioned above drugs alone,the inhibition of proliferation potenital and apoptosis rate of cells in combination group markedly increase with time and concentration dependent marner( P < 0. 01),the exprssion of NF-κB,BCL-2 and TGF-β mRNA and proteins decreased. Conclusion: Emodin combined AZT can synergistically inhibit the proliferation,induce cell apoptosis,and down regulate the expression of NF-κB 、BCL-2 and TGF-β mRNA and proteins in the CLSC,the possible mechanism of synergistic effect may be associated with inhibiton of BCL-2 activation and down-regulation of the expression of NF-κB,and TGF-β.