全反式维甲酸对小鼠黑色素瘤B16F10细胞生物学行为的影响

发布时间：2018-03-09 08:23

本文选题：全反式维甲酸　切入点：恶性黑色素瘤　出处：《第三军医大学学报》2017年22期 　论文类型：期刊论文

【摘要】：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(P0.05);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(P0.05);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(P0.05);成瘤实验结果显示ATRA处理后的B16F10细胞致瘤性显著降低(P0.01),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。
[Abstract]:Objective to investigate the proliferation, migration and apoptosis of mouse melanoma B16F10 cells induced by all-trans retinoic ATRAA. Methods the effects of ATRA on the proliferation of mouse melanoma cells B16F10 and mouse fibroblasts L929 were detected by MTT method. The morphological changes of the two cells were observed under microscope. The effect of ATRA on the migration of B16F10 cells was detected by scratch test and Transwell assay. The apoptosis of B16F10 cells was observed by DAPI staining and Annexin V-FITC / Pi staining. The mitochondrial membrane potential of B16F10 cells was detected by JC-1 staining. The activation of Caspase-9 and Caspase-3 in B16F10 cells was detected by fluorescence spectrophotometer, and the effect of ATRA on the tumorigenicity of B16F10 cells was observed by in vivo tumorigenesis. Results MTT showed that ATRA inhibited the proliferation of B16F10 cells and L929 cells in a dose-dependent manner. However, the survival rate of B16F10 cells was significantly lower than that of L929 cells (P 0.05). After treated with ATRA at low, medium and high concentrations of 6.25 ~ 1640 渭 mol / L for 24 h, it was observed that B16F10 cells showed apoptosis-like changes at medium concentration and cell death at high concentration. L929 cells lost normal morphology, while L929 cells only changed significantly at high concentration. The results of scratch test and Transwell assay showed that the migration ability of B16F10 cells in vitro was significantly inhibited at medium concentration (P 0.05), and nuclear cleavage could be observed by 24 h DAPI staining of B16F10 cells treated with ATRA at medium concentration of 16 渭 mol / L. Even the apoptotic body Annexin V-FITC / Pi staining combined with flow cytometry showed that the apoptosis rate was 37.27 卤4.42% and the mitochondrial membrane potential was significantly decreased, and Caspase-9 caspase-3 was activated by fluorescence spectrophotometer. The results of tumorigenesis experiment showed that the tumorigenicity of B16F10 cells treated with ATRA significantly decreased or even disappeared. Conclusion ATRA can significantly inhibit the proliferation and migration of B16F10 cells in vitro, and can decrease mitochondrial membrane potential. Activation of Caspase-9 and Caspase-3 protein induces apoptosis and inhibits B 16 F 10 tumorigenicity.
【作者单位】：重庆医科大学附属大学城医院药学部;第三军医大学大坪医院野战外科研究所药剂科;第三军医大学药学院教学实验中心;
【分类号】：R965

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