汉防己甲素对人多发性骨髓瘤干细胞增殖、凋亡的影响及机制

Effect and Mechanism of Tetrandrine on Proliferation and Apoptosis in Multiple Myeloma Stem Cells

目的:研究汉防己甲素对人多发性骨髓瘤干细胞CD138^- B细胞增殖、凋亡的影响及作用机制。方法:利用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)比色法检测汉防己甲素对CD138^- B细胞增殖的影响;应用流式细胞仪检测汉防己甲素对CD138^- B细胞周期的影响以及药物诱导细胞凋亡的作用;应用蛋白免疫印迹法(Western blot)检测汉防己甲素对CD138^- B细胞中裂解的半胱氨酸天冬氨酸蛋白水解酶(cleaved-Caspase)-3,cleaved-Caspase-9蛋白表达的影响。结果:与空白组及溶剂组比较,10,20,30,40μmol·L^(-1)汉防己甲素处理6,12,24 h对CD138^- B细胞的增殖均具有显著的抑制作用(P<0.01);与空白组及溶剂组比较,汉防己甲素30,40μmol·L^(-1)时,G_0/G_1期细胞显著增多(P<0.01),S期细胞明显减少(P<0.05,P<0.01),汉防己甲素10,20,30,40μmol·L^(-1)时,细胞凋亡率显著增加(P<0.01);与空白组及溶剂组比较,20,30,40μmol·L^(-1)汉防己甲素处理后,cleaved-Caspase-3,cleaved-Caspase-9蛋白表达显著升高(P<0.01),10μmol·L^(-1)汉防己甲素处理时,cleaved-Caspase-3蛋白表达明显增高(P<0.05)。结论:汉防己甲素通过阻滞细胞周期在G_0/G_1期抑制CD138^- B细胞的增殖,通过活化细胞内与凋亡相关蛋白cleaved-Caspase-3,cleaved-Caspase-9促进细胞凋亡。

Objective： To investigate the effects and mechanism of tetrandrine on proliferation and apoptosis in multiple myeloma stem cells. Method： Methylthiazolyldiphenyl-tetrazolium bromide（MTT） assay was used to detect the survival of cancer cell. Flow cytometer was used to detect the cell circles and apoptotic induced by tetrandrine. Western blot were used to detect the activity of cleaved-Caspase-3,cleaved-Caspase-9. Result：Compared with blank group and solution group,10,20,30,40 μmol·L-1tetrandrine treatment for 6,12,24 h could significantly inhibit the proliferation of CD138- B cells（P〈0.01）. Compared with blank group and solution group,the cells in G0/G1 phase increased significantly（P〈0.01）,and the cells in S phase significantly decreased（P〈0. 05,P〈0.01）,when being treated with 30,40 μmol·L-1tetrandrine. The apoptotic rate increased significantly at concentrations of 10,20,30,40 μmol·L-1of tetrandrine（P〈0.01）. Compared with blank group and solution group,the expression of cleaved-Caspase-3,cleaved-Caspase-9 protein was significantly increased after being treated with 20,30,40 μmol·L-1tetrandrine（P〈0.01）,the expression of cleavedCaspase-3 protein was significantly increased when being treated with 10 μmol·L-1tetrandrine（P〈0. 05）.Conclusion： Tetrandrine can arrest CD138^- B cell line in the G0/G1 phase,inhibit the cell survival,and enhance CD138^- B cell apoptosis by promoting the activity of cleaved-Caspase-3,cleaved-Caspase-9.