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目的阿帕替尼(Apatinib)为新一代小分子血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2,VEGFR-2)酪氨酸激酶抑制剂,对多种实体肿瘤细胞有杀伤作用。本研究探讨Apatinib对急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)细胞株Nalm6增殖抑制及诱导凋亡的作用。方法 CCK8法检测不同浓度Apatinib对Nalm6细胞的增殖抑制作用,Annexin V/PI法检测不同浓度Apatinib诱导Nalm6细胞的凋亡情况,蛋白质印迹法法检测Apatinib处理后Bcl-2、Bcl-xL和PARP蛋白的表达变化。结果 CCK-8细胞毒性实验结果显示,Apatinib对Nalm6细胞具有明显的增殖抑制作用,呈剂量及时间依赖性,作用48h后,2.5、5、10、20和40μmol/L的Apatinib对Nalm6细胞的增殖抑制率分别为(3.78±1.01)%、(13.36±1.42)%、(25.80±2.83)%、(44.40±7.74)%和(64.07±6.66)%,经多个独立样本非参数检验Kruskal-Wallis H分析,各浓度的Apatinib均能诱导Nalm6细胞凋亡,与对照组(3.61±0.91)%比较差异有统计学意义,χ~2=13.500,P=0.009;作用72h后,2.5、5、10、20和40μmol/L的Apatinib对Nalm6细胞的增殖抑制率分别为(4.57+2.38)%、(20.26+4.06)%、(39.27±1.57)%、(65.19±4.45)%和(85.54±3.37)%,经检验各浓度的Apatinib均能抑制Nalm6细胞增殖,与对照组比较差异有统计学意义,χ~2=13.500,P=0.009。48和72h的IC50分别为(24.39±0.05)和(13.18±0.08)μmol/L。凋亡实验显示,Apatinib能增加Nalm6细胞凋亡率,并呈剂量依赖性,作用48h后,对照组和10、20、30、40μmol/L Apatinib组对Nalm6细胞的凋亡率分别为(3.61±0.91)%、(5.28±1.29)%、(5.9±0.85)%、(10.18±1.48)%和(15.23±3.69)%,经检验各浓度的Apatinib均能诱导Nalm6细胞凋亡,与对照组比较差异有统计学意义,χ~2=12.433,P=0.014;作用72h后,对照组和10、20、30、40μmol/L Apatinib组对Nalm6细胞的凋亡率分别为(3.8±1.10)%、(5.33±2.08)%、(10.10±2.86)%、(12.15±1.43)%和(25.61±3.63)%,经检验各浓度的Apatinib均能诱导Nalm6细胞凋亡,与对照组比较差异有统计学意义,χ~2=12.233,P=0.016。蛋白质印迹法结果显示,Apatinib作用Nalm6细胞24、48h后均能下调Bcl-2和Bcl-xl的表达,切割PARP蛋白。结论 Apatinib能抑制Nalm6细胞增殖,并诱导其凋亡,其机制可能与下调Bcl-2和Bcl-xl有关。
Purpose Apatinib is a new generation of small molecule inhibitor of tyrosine kinase of vascular endothelial growth factor receptor-2 (VEGFR-2), which can kill many kinds of solid tumor cells . This study was designed to investigate the effect of Apatinib on proliferation and apoptosis of acute lymphoblastic leukemia (ALL) cell line Nalm6. Methods CCK8 was used to detect the proliferation of Nalm6 cells induced by different concentrations of Apatinib. The apoptosis of Nalm6 cells induced by different concentrations of Apatinib was detected by Annexin V / PI. The protein expressions of Bcl-2, Bcl-xL and PARP were detected by Western blotting Changes in expression. Results The cytotoxicity of CCK-8 showed that Apatinib had a significant inhibitory effect on the proliferation of Nalm6 cells in a dose- and time-dependent manner. After 48h, the proliferation of Nalm6 cells induced by Apatinib at 2.5, 5, 10, 20 and 40μmol / L The inhibitory rates were (3.78 ± 1.01)%, (13.36 ± 1.42)%, (25.80 ± 2.83)%, (44.40 ± 7.74)% and (64.07 ± 6.66)% respectively. Kruskal-Wallis H analysis showed that all the concentrations of Apatinib could induce the apoptosis of Nalm6 cells compared with the control group (3.61 ± 0.91)%, the difference was statistically significant, χ ~ 2 = 13.500, P = 0.009; after 72h, 2.5,5,10 (4.57 ± 2.38)%, (20.26 ± 4.06)%, (39.27 ± 1.57)%, (65.19 ± 4.45)% and (85.54 ± 3.37)% respectively for Apatinib and 20 and 40μmol / %. The tested Apatinib at various concentrations inhibited the proliferation of Nalm6 cells with statistical significance compared with the control group. The IC50 of χ ~ 2 = 13.500, P = 0.009.48 and 72h were (24.39 ± 0.05) and (13.18 ± 0.08) μmol / L. Apoptosis experiments showed that Apatinib could increase the apoptosis rate of Nalm6 cells in a dose-dependent manner. The apoptotic rate of Nalm6 cells in control and 10, 20, 30, 40μmol / L Apatinib groups after 48h treatment were (3.61 ± 0.91%, 5.28 ± 1.29%, 5.9 ± 0.85%, 10.18 ± 1.48% and 15.23 ± 3.69% respectively.Apoptosis of Nalm6 cells was induced by various concentrations of Apatinib compared with the control group The difference was statistically significant (χ ~ 2 = 12.433, P = 0.014). The apoptotic rate of Nalm6 cells in control and 10, 20, 30, 40μmol / L Apatinib groups were (3.8 ± 1.10)% (5.33 ± 2.08)%, (10.10 ± 2.86)%, (12.15 ± 1.43)%, and (25.61 ± 3.63)%, respectively. All the tested concentrations of Apatinib could induce the apoptosis of Nalm6 cells compared with the control group Significance, χ ~ 2 = 12.233, P = 0.016. Western blotting showed that Apatinib could down-regulate the expression of Bcl-2 and Bcl-xl, and cleave PARP protein 24 and 48 h after Nalm6 treatment. Conclusion Apatinib can inhibit the proliferation and induce the apoptosis of Nalm6 cells, which may be related to the down-regulation of Bcl-2 and Bcl-xl.