全反式维甲酸对小鼠黑色素瘤B16F10细胞生物学行为的影响

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、40 μmol/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(16 μmol/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;AnnexinV-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.

Effects of all-trans retinoic acid on biological behaviors in murine melanoma cell line B16F10

Objective To investigate the inhibitory effects of all-trans retinoic acid (ATRA) on the proliferation,migration,apoptosis and tumorigenicity of B16F10 cells and the preliminary mechanism in vitro and in vivo.Methods The effect of ATRA on the proliferation of murine melanoma cell line B16F10 and mouse fibroblast cell line L929 was detected with MTT assay.The morphological changes were observed in the 2 cell lines after treatment under an inverted microscope.Migration ability of B16F10 cells after ATRA treatment was measured by wound healing assay and Transwell chamber assay.The apoptosis of B16F10 cells was analyzed with the use of DAPI staining and Annexin V-FITC/PI staining.The effect of ATRA on the mitochondrial membrane potential and the expression of Caspase-9 and Caspase-3 in the B16F10 cells were detected by JC-1 staining and fluorescence spectrophotometry respectively.Tumorigenicity in vivo was evaluated by tumor formation in C57BL/6 mice.Results ATRA exerted inhibitory effects on the proliferation in B16F10 and L929 cells in dose-dependent manners,but the survival rate was significantly lower in the B16F10 cells than the L929 cells (P ＜ 0.05).After the 2 cell lines were treated with ATRA at low,moderate and high doses (6.25,16 and 40 μmol/L) respectively for 24 h,the B16F10 cells displayed apoptosis-like changes under moderate dose,and almost all died under high dose,without normal cellular morphology,while,the L929 cells only had morphological changes under high dose of ATRA.Wound healing and Transwell chamber test showed that moderate dose of ATRA (16 μmol/L) significantly inhibited the migration and invasion abilities in B16F10 cells,and the treatment for 24 h induced cell apoptosis,confirmed by nuclear cleavage and apoptotic bodies after DAPI staining,with a apoptotic rate of (37.27 ± 4.42) ％.The mitochondrial membrane potential was reduced while the expression levels of Caspase-9 and Caspase-3 were increased by 1.54 ±0.00 and 3.09 ± 0.01 times respectively (P ＜ 0.05).ATRA treatment also resulted in decreased and even no tumorigenicity of B16F10 cells (P ＜ 0.001).Conclusion ATRA treatment significantly decreases the proliferation and migration of B16F10 cells,and further inhibits tumorigenicity through decreasing mitochondrial potential and activating Caspase-9 and Caspase-3 to promote cell apoptosis.