低浓度雷公藤内酯联合伊达比星在急性髓系白血病干细胞凋亡中的作用

目的 研究IC20浓度(5 nmoL/L)的雷公藤内酯(TPL)联合IC50浓度(27 nmol/L)的伊达比星(IDA)能否通过下调MDR1基因表达水平诱导急性髓系白血病(AML)干细胞凋亡.方法 从KG1a细胞株中分选CD34+ CD38-亚群,将其作为AML干细胞模型;分成空白对照组、TPL组、IDA组、联合组.空白对照组:培养液中不合TPL及IDA;TPL组:培养液中含IC20浓度(5 nmoL/L)的TPL;IDA组:培养液中合IC50浓度(27 nmoL/L)的IDA;联合组:培养液中含IC20浓度(5 nmoL/L)的TPL及IC50浓度(27 nmoL/L)的IDA.Annexin V-FITC/PI双标法流式细胞术检测处理后AML干细胞的凋亡情况;RT-PCR法检测上述4组中AML干细胞MDR1 mRNA表达水平的变化;Western blot检测上述4组AML干细胞给药后MDR1蛋白表达水平的变化.结果 空白对照组、TPL组、IDA组及联合组中AML干细胞在处理24 h后凋亡率分别为(0.37±0.29)％、(18.07±0.32)％、(20.13±0.06)％和(60.17±1.45)％,联合组中AML干细胞凋亡比例显著高于TPL组及IDA组,差异有统计学意义(P＜0.001).RT-PCR结果显示TPL组、IDA组及联合组中AML干细胞内MDR1 mRNA表达水平均有不同程度下调,空白对照组、TPL组、IDA组及联合组中MDR1 mRNA表达水平分别为0.927±0.084、0.462±0.048、0.396±0.019和0.231±0.023,联合组MDR1 mRNA表达水平均显著低于TPL组及IDA组,差异有统计学意义(P＜0.001).Western blot结果显示TPL组、IDA组及联合组AML干细胞内MDR1蛋白表达水平均不同程度下调,其中联合组更加明显.结论 IC20浓度的TPL可显著提高IC50浓度的IDA对AML干细胞的凋亡诱导作用,其机制可能与抑制MDR1基因及蛋白的表达有关.

Low-dose triptolide in combination with idarubicin induces apoptosis in acute myeloid leukemia stem cells

Objective To investigate the role of MDR1 in the cytotoxicity of low-dose tripelide (TPL) in combination with Idarubicin (IDA) on acute myeloid leukemia (AML) stem cells.Methods CD34 + CD38-KG1a cells sorted from KG1a cell line by MACS were subjected to TPL alone,IDA alone and their combination for 24 hours.The apoptotic rate of CD34 + CD38-KG1a cells was determined by flow cytometry with Annexin V/PI double staining.The mRNA expression of MDR1 was measured by SYBR RT-PCR.Western blot analysis was performed to detect the protein expression of MDRI.Results After exposure to 5 nmol/L TPL (IC20) alone,27 nmol/L IDA (IC50) alone,and 5 nmol/L TPL with 27 nmol/L IDA,the apoptotic rates of CD34 + CD38-KG1 a cells were (18.07 ± 0.32) ％,(20.13 ± 0.06) ％,and (60.17 ± 1.45)％,respectively.The combination of TPL and IDA-inducing apoptosis was significantly increased compared to IDA or TPL alone.According to RT-PCR results,the mRNA expression of MDR1 could be reduced by TPL alone,IDA alone and their combination.Compared to the control group (0.927 ± 0.084),the relative expression of MDR1 was 0.462 ±0.048,0.396 ± 0.019,and 0.231 ± 0.023,respectively.A remarkable suppression of MDR1 was observed in the combination group.Western blot results further confirmed a marked inhibition of MDRI in the combination group.Conclusion Low-dose TPL significantly enhanced the cytotoxicity of IDA in AML stem cells via inhibition of MDR1 expression.