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目的探讨全反式维甲酸(all-trans retinoic acid,ATRA)对小鼠黑色素瘤B16F10细胞体外增殖、迁移、凋亡、致瘤性的影响及其初步作用机制。方法采用MTT法检测ATRA对小鼠黑色素瘤细胞B16F10和小鼠成纤维细胞L929增殖能力的影响。显微镜下观察给药后两种细胞的形态变化,应用划痕实验和Transwell实验检测ATRA对B16F10细胞迁移能力的影响,分别采用DAPI染色和Annexin V-FITC/PI染色观察B16F10细胞凋亡情况,JC-1染色实验检测B16F10细胞线粒体膜电位,荧光分光光度计检测B16F10细胞中Caspase-9、Caspase-3的活化情况,利用体内成瘤实验观察ATRA对B16F10细胞致瘤性的影响。结果 MTT结果显示ATRA呈浓度依赖性地抑制B16F10细胞和L929细胞增殖,但B16F10细胞的存活率明显低于L929细胞的存活率(P<0.05);分别以低、中、高3个浓度(6.25、16、40μmol/L)的ATRA处理细胞24 h后,显微镜下可见B16F10细胞在中浓度时呈凋亡样改变,高浓度时细胞基本死亡,丧失正常形态;而L929细胞形态仅在高浓度时发生显著变化;划痕实验和Transwell实验提示B16F10细胞的体外迁移能力在中浓度即受到明显抑制(P<0.05);继续以中浓度(16μmol/L)的ATRA作用B16F10细胞24 h,DAPI染色可见细胞核裂解,甚至可见凋亡小体;Annexin V-FITC/PI染色结合流式细胞仪测得细胞凋亡率为(37.27±4.42)%;JC-1染色实验提示线粒体膜电位明显降低;荧光分光光度计测得给药后Caspase-9、Caspase-3显著被活化,活性分别增加了(1.54±0.00)、(3.09±0.01)倍(P<0.05);成瘤实验结果显示ATRA处理后的B16F10细胞致瘤性显著降低(P<0.01),甚至消失。结论 ATRA能显著抑制B16F10细胞体外增殖、迁移能力,并能通过降低线粒体膜电位,活化Caspase-9、Caspase-3蛋白诱导细胞凋亡,进而抑制B16F10致瘤性。
Objective To investigate the effect of all-trans retinoic acid (ATRA) on the proliferation, migration, apoptosis and tumorigenicity of mouse melanoma B16F10 cells and its primary mechanism. Methods MTT assay was used to detect the effect of ATRA on the proliferation of mouse melanoma B16F10 and mouse fibroblast L929. Morphological changes of the two kinds of cells were observed under microscope. Scratch assay and Transwell assay were used to detect the effect of ATRA on the migration of B16F10 cells. DAPI staining and Annexin V-FITC / PI staining were used to observe the apoptosis of B16F10 cells. JC -1 staining was used to detect the mitochondrial membrane potential of B16F10 cells. The activation of Caspase-9 and Caspase-3 in B16F10 cells was detected by fluorescence spectrophotometer. The in vivo tumorigenicity assay was used to observe the effect of ATRA on the tumorigenicity of B16F10 cells. Results MTT assay showed that ATRA inhibited the proliferation of B16F10 and L929 cells in a concentration-dependent manner, but the survival rate of B16F10 cells was significantly lower than that of L929 cells (P <0.05) , 16,40 micromol / L) ATRA for 24 h, B16F10 cells showed apoptosis-like changes at medium concentration, and the cells died at high concentration, losing their normal morphology. However, the morphology of L929 cells was only at high concentration (P <0.05). The proliferation of B16F10 cells was inhibited by ATRA in medium concentration (16μmol / L) for 24 h and DAPI staining was observed (37.27 ± 4.42)%. Annexin V-FITC / PI staining combined with flow cytometry showed that the mitochondrial membrane potential was significantly decreased by JC-1 staining. Fluorescence spectroscopy After photodynamic therapy, the activity of Caspase-9 and Caspase-3 were significantly increased (1.54 ± 0.00) and (3.09 ± 0.01) times respectively (P <0.05). The results of tumorigenesis showed that B16F10 Cell tumorigenicity was significantly lower (P <0.01), or even disappearConclusions ATRA can significantly inhibit the proliferation and migration of B16F10 cells in vitro and induce apoptosis of B16F10 cells by decreasing the mitochondrial membrane potential and activation of Caspase-9 and Caspase-3 proteins, thereby inhibiting the tumorigenicity of B16F10.