雷帕霉素对人急性髓系白血病HL-60细胞凋亡及Bax、Bcl-2表达的影响

摘要

Objective To investigate the effect of rapamycin on apoptosis of HL- 60 cells and the expression of Bax and Bcl-2 in patients with human acute myeloid leukemia. Methods Human HL-60 cells were cultured in vitro and treated with different concentrations of rapamycin (10 nmol/L, 20 nmol/L, 40 nmol/L, 80 nmo)/L, 160 nmol/L), along with blank control group and the same volume of DMSO group. CCK8 was used to detect the cell proliferation inhibition rate. The apoptotic cells were observed by fluorescence microscope and TdT- mediated terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. Then the expression of Bax and Bcl-2 at mRNA and protein levels were detected by QPCR and western blot in 48 hours. Results CCK- 8 assay results showed that the growth inhibition rate of rapamycin treatment group was significantly higher than that in control group and DMSO group (P ＜0.05), and the inhibition rate was also increased with the increase of concentration. With the increase of rapamycin concentration, the apoptotic rate of HL- 60 cells was (8.4±1.3)％, (14.7土1.9)％,(28.5±2.7)％, (51.6 ±5.4)％, (52.7 ±4.8)％, respectively. Which were significantly different from those of the control group (P ＜0.01 ). Compared with the control group, the mRNA and protein expression of Bax increased with the increase of rapamycin concentration (P ＜0.01). However, the expression of Bcl-2 at mRNA and protein level decreased (P ＜0.01). Conclusion Rapamycin can inhibit the growth of HL- 60 cells and may induce HL- 60 cell apoptosis by promoting the expression of Bax and inhibiting the production of Bcl- 2, and the above effects are dose dependent.%目的 探讨雷帕霉素对人急性髓系白血病HL-60细胞凋亡及Bax、Bcl-2表达的影响.方法 体外培养人 HL-60细胞,并将不同浓度(10nmol/L、20nmol/L、40nmol/L、80nmol/L、160nmol/L) 雷帕霉素(溶于 DMSO)作用于HL-60细胞,另设空白对照组和相同体积的DMSO组.然后应用CCK8技术检测各组细胞增殖抑制率,应用荧光显微镜和TdT酶介导的末端缺失原位标记法观察其凋亡情况,应用QPCR、Western blot等方法观察48h不同组别Bax、Bcl-2基因和蛋白表达情况.结果 CCK-8检测结果显示,不同浓度雷帕霉素处理组细胞生长抑制率明显大于对照组和 DMSO组(P＜0.05),而且随着浓度的增加,其抑制率也明显增高.随着雷帕霉素浓度的增加,HL-60凋亡细胞逐渐增多,细胞凋亡率分别为(8.4±1.3)％、(14.7±1.9)％、(28.5±2.7)％、(51.6±5.4)％、(52.7±4.8)％,与对照组相比差异均有统计学意义(P＜0.01).QPCR和Western blot结果显示,和对照组相比,随着雷帕霉素浓度的增加BaxmRNA和蛋白表达水平逐渐增高,且均远大于对照组(P＜0.01);然而Bcl-2mRNA和蛋白表达水平却随着雷帕霉素浓度的增加逐渐降低,且均远小于对照组(P＜0.01).结论 雷帕霉素可以明显抑制HL-60细胞生长,并可能通过促进Bax表达,抑制Bcl-2生成来诱导HL-60细胞凋亡,且上述作用呈剂量依赖性.