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目的探讨达沙替尼对小鼠黑色素瘤B16F10细胞株体外增殖、迁移和凋亡的影响。方法采用MTT法检测不同浓度(3.125、6.250、12.500、25.000、50.000μg/m L)达沙替尼在24、48 h对B16F10细胞增殖能力的影响;采用细胞划痕实验和Transwell体外迁移实验检测达沙替尼对B16F10细胞迁移能力的影响;DAPI染色法观察给药后细胞核形态的变化;流式细胞仪检测达沙替尼对B16F10细胞凋亡率和细胞周期的影响;荧光显微镜观察达沙替尼对B16F10细胞线粒体膜电位的影响,并用荧光分光光度计检测Caspase 3和Caspase 9的活化程度。结果达沙替尼对B16F10细胞的增殖有抑制作用,并呈浓度和时间依赖性。细胞划痕实验及Transwell体外迁移实验结果表明:达沙替尼能够有效地抑制B16F10细胞的迁移。分别以低、中、高3个浓度(6.250、12.500、25.000μg/m L)的达沙替尼作用于B16F10细胞24 h后,细胞形态发生明显改变,核裂解,形成多个凋亡小体,凋亡率分别为(34.06±0.83)%、(50.24±1.66)%和(88.91±0.96)%,与对照组比较差异均有统计学意义(P<0.05)。达沙替尼对B16F10细胞周期的影响较小,其中对G1期有微弱的阻滞作用。中浓度(12.500μg/m L)达沙替尼组能够引起B16F10细胞线粒体膜电位的降低,Caspase 3和Caspase 9活性的增加,表明达沙替尼可能是通过线粒体介导的Caspase通路引起细胞凋亡。结论达沙替尼能有效地抑制小鼠黑色素瘤B16F10细胞株体外增殖与迁移,诱导细胞凋亡,有望成为一种有效的抗黑色素瘤药物。
Objective To investigate the effects of dasatinib on the proliferation, migration and apoptosis of murine melanoma B16F10 cells in vitro. Methods The proliferation of B16F10 cells was detected by MTT assay at different concentrations (3.125, 6.250, 12.500, 2.50, 5.000 μg / mL) for 24 h and 48 h. Dasatinib was tested by cell scratch assay and Transwell migration assay Dasatinib on the migration of B16F10 cells; DAPI staining observed after administration of nuclear morphology changes; Dasatinib by flow cytometry B16F10 apoptosis rate and cell cycle; Fluorescence microscopy Dasha The effect of tenini on the mitochondrial membrane potential of B16F10 cells was examined. The activation of Caspase 3 and Caspase 9 were detected by fluorescence spectrophotometer. Results Dasatinib inhibited the proliferation of B16F10 cells in a concentration- and time-dependent manner. Cell scratch assay and in vitro transwell migration assay showed that dasatinib can effectively inhibit the migration of B16F10 cells. Drosatinib at low, medium and high concentrations (6.250,12.500,25.000μg / m L) were applied to B16F10 cells for 24 h, respectively. The morphological changes of cells were observed and nuclear fragmentation was formed, leading to the formation of multiple apoptotic bodies (34.06 ± 0.83)%, (50.24 ± 1.66)% and (88.91 ± 0.96)%, respectively, which were significantly different from the control group (P <0.05). Dasatinib had little effect on the cell cycle of B16F10 cells, but had a weak block on G1 phase. Dasatinib at a concentration of 12.500μg / mL could induce a decrease in mitochondrial membrane potential and an increase in the activity of Caspase 3 and Caspase 9 in B16F10 cells, indicating that dasatinib may induce apoptosis through mitochondria-mediated Caspase pathway Death. Conclusion Dasatinib can effectively inhibit the proliferation and migration of mouse melanoma cell line B16F10 and induce cell apoptosis in vitro. Dasatinib is expected to become an effective anti-melanoma drug.