摘要目的 研究吉西他滨联合ABT-199对费城染色体阳性(Ph+)急性淋巴细胞白血病(ALL)细胞株SUP-B15的增殖抑制及凋亡诱导作用,并探讨其协同作用机制.方法 取对数生长期SUP-B15细胞,分别接受吉西他滨(0.025、0.050μmol/L)、ABT-199(0、0.5、1.0、2.0、4.0、8.0μmol/L)及两药联合处理24 h后,采用CCK-8法检测细胞增殖情况,流式细胞术(FCM)检测细胞凋亡情况,JC-1法检测线粒体膜电位的变化,蛋白质印迹法分析线粒体凋亡通路相关蛋白的表达变化.结果 ABT-199作用SUP-B15细胞24 h的半数抑制浓度(IC50)值为(4.13±0.89)μmol/L,吉西他滨(0.025、0.050μmol/L)能增强ABT-199对SUP-B15细胞的增殖抑制作用,其IC50值分别为(2.23±0.73)、(1.15±0.45)μmol/L,差异均有统计学意义(t=28.65,P<0.01;t=35.12,P<0.01).FCM检测细胞凋亡结果显示,与0.025μmol/L吉西他滨单药组[(7.33±1.54)％]相比,0.025μmol/L吉西他滨联合ABT-199(1.0、2.0μmol/L)作用SUP-B15细胞24 h后,凋亡细胞比例分别为(32.42±1.45)％和(44.33±1.86)％,差异具有统计学意义(F=70.78,P<0.001);与0.050μmol/L吉西他滨单药组[(9.60±2.76)％]相比,0.050μmol/L吉西他滨联合ABT-199(1.0、2.0μmol/L)后,凋亡细胞比例增加,分别为(47.63±3.81)％和(58.73±4.33)％,差异有统计学意义(F=79.21,P<0.001).吉西他滨联合ABT-199作用SUP-B15细胞12 h后,去极化细胞的比例高于单药组,差异有统计学意义(P<0.001).吉西他滨联合ABT-199作用SUP-B15细胞12 h后,抗凋亡蛋白bcl-2、bcl-xL、Mcl-1水平下降.结论 吉西他滨可增强ABT-199对Ph+ALL细胞的增殖抑制及凋亡诱导作用,机制可能与下调抗凋亡相关蛋白相关.

abstractsObjective To investigate the effects of gemcitabine and ABT-199 on proliferation inhibition and apoptosis induction of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) cell line SUP-B15, and to explore its synergistic mechanism. Methods SUP-B15 cells in logarithmic growth phase were treated with gemcitabine (0.025 and 0.050 μmol/L), ABT-199 (0, 0.5, 1.0, 2.0, 4.0, 8.0 μmol/L) or two drugs for 24 h. Cell proliferation was detected by CCK-8 method, apoptosis was detected by flow cytometry (FCM), mitochondrial membrane potential was detected by JC-1 method, and expression of mitochondrial apoptosis pathway-related protein was analyzed by Western blot. Results The 50 ％ inhibitory concentration (IC50) of SBT-B15 cells treated with ABT-199 for 24 h was (4.13±0.89) μmol/L. However, gemcitabine (0.025, 0.050 μmol/L) significantly enhanced the inhibitory effect of ABT-199 on proliferation of SUP-B15 cells, the IC50 values were (2.23 ±0.73) and (1.15 ±0.45) μmol/L, respectively. The results of FCM assay showed that compared with the monotherapy group [(7.33±1.54)％], 0.025 umol/L gemcitabine combined with ABT-199 (1.0 and 2.0 μmol/L) acted on SUP-B15 cells for 24 h, the proportions of apoptotic cells were (32.42±1.45) ％and (44.33±1.86) ％, the difference was statistically significant (F=70.78, P<0.001);compared with the monotherapy group [(9.60 ±2.76) ％], 0.05 μmol/L gemcitabine combined with ABT-199 (1.0 and 2.0 μmol/L) acted on SUP-B15 cells for 24 h, the proportion of apoptotic cells increased to (47.63 ± 3.81) ％ and (58.73 ±4.33) ％, respectively, and the difference was statistically significant (F= 79.21, P<0.001). The JC-1 experiment showed that treated with ABT-199 and gemcitabine for 12 h, the percentage of depolarizing cell was significantly higher than that in single agent group, and the difference was statistical significant (P<0.001). Western blot showed that the anti-apoptotic proteins bcl-2, bcl-xL and Mcl-1 decreased after treated by gemcitabine combined with ABT-199 for 12 h. Conclusion Gemcitabine could enhance the proliferation inhibition and induce apoptosis of Ph+ALL cells by ABT-199, and its mechanism may be related to down-regulation of anti-apoptosis-related proteins.