重组人血小板生成素对急性髓系白血病细胞株增殖及凋亡的影响

Effect of rhTPO to the Proliferation and Apoptosis of Acute Myeloid Leukemia Cell Lines

目的:探索重组人血小板生成素(rh TPO)对急性髓系白血病(AML)细胞株增殖和凋亡的影响。方法:采用CCK-8法检测AML细胞株(Kasumi-1、Skno-1、HEL、HL-60、THP-1)经不同浓度rh TPO(0、50、100 ng/ml)作用不同时间(24、48、72 h)后的细胞增殖率;Annexin V/PI法检测不同浓度rh TPO处理后各AML细胞株的细胞凋亡率;QPCR法检测各AML细胞株的TPO受体c-MPL(myeloproliferative leukemia)mRNA的相对表达量;Western blot法检测各AML细胞株的c-MPL蛋白质表达量;流式细胞术检测不同浓度rh TPO处理后HL-60细胞c-MPL抗原表达量的变化。结果:rh TPO对Kasumi-1、Skno-1、HEL、HL-60、THP-1细胞均无促进增殖作用,然而在HL-60细胞(72 h 0vs 100 ng/ml,P=0.008)中呈现细胞增殖的抑制效应及促凋亡效应(48 h 0 vs 50 ng/ml,P=0.0143)。在Kasumi-1、Skno-1、HEL和THP-1细胞中,经不同浓度rh TPO处理后各组间的细胞凋亡率无统计学差异。各AML细胞株均有不同程度的c-MPL基因和c-MPL蛋白质表达,其中,HEL细胞中两者表达量最高。不同浓度rh TPO处理HL-60细胞48 h后,各组间的c-MPL抗原表达量无统计学差异。结论:rh TPO在体外对Kasumi-1、Skno-1、HEL、HL-60和THP-1白血病细胞系无促增殖作用。相反,rh TPO可以抑制HL-60细胞增殖、促进其凋亡,而这种效应与c-MPL基因及其蛋白质表达均无关。

Objective:To investigate the effects of recombinant human thrombopoietin(rhTPO)to proliferation and apoptosis of acute myeloid leukemia(AML)cell lines.Methods:After the treatment of different concentrations of rhTPO(0,50,100 ng/ml)for different time(24,48,72 h),the cell proliferation rates of the AML cell lines(Kasumi-1,Skno-1,HEL,HL-60,THP-1)were determined by CCK-8 method.Apoptosis rate of each cell line cocultured with rhTPO was detected by Annexin V/PI method.The relative expression of TPO receptor c-MPL(myeloproliferative clonal antibody)mRNA in AML cell lines was detected by Q-PCR.The expression of c-MPL protein in each cell line was detected by Western blot.The expression of c-MPL antigen in HL-60 cells treated by different concentrations of rhTPO was detected by Flow cytometry.Results:RhTPO showed no promotion to the proliferation of Kasumi-1,Skno-1,HEL,HL-60,THP-1 cell lines,however,it showed inhibitory effect to cell proliferation(72 h 0 ng/ml vs 100 ng/ml,P=0.029)and pro-apoptotic(48 h 0 ng/ml vs 50 ng/ml,P=0.0143)in HL-60 cells.In Kasumi-1,Skno-1,HEL and THP-1 cells,there showed no statistically significant differences in apoptosis rate among each groups treated by different concentrations of rhTPO.Each AML cell line showed different levels of c-MIPL gene and c-MPL protein expression,but HEL cells showed the highest expression in both of them.After HL-60 cells were treated by different concentrations of rhTPO for 48 hours,there showed no statistical difference in c-MPL antigen expression among each groups.Conclusion:RhTPO can not promote the proliferation of Kasumi-1,Skno-1,HEL,HL-60 and THP-1 leukemia cell lines.On the contrary,rhTPO can inhibit HL-60 cell proliferation and promote its apoptosis,and this effect is not related to c-MPL gene expression or protein expression.