相思子蛋白P2对B16黑色素瘤的抑制作用及机制研究

The inhibition of abrin P2 to B16 melanoma and its mechanism

目的:研究相思子蛋白P2对小鼠黑色素瘤B16生长的抑制作用及作用机制。方法:MTT法观察相思子蛋白P2对B16细胞增殖的抑制作用,流式细胞仪检测对B16细胞周期和细胞凋亡的影响,JC-1染色法检测相思子蛋白P2对B16细胞线粒体膜电位的影响;相思子蛋白P2 50、75、100μg/kg连续灌胃给药10天,观察对小鼠黑色素瘤B16移植性肿瘤生长的抑制作用。结果:相思子蛋白P2对B16细胞具有显著的生长抑制作用,IC50值为4.6×10-3μg/ml,浓度为1μg/ml时抑制率可达95.45%;流式细胞仪分析显示,相思子蛋白P2主要将细胞周期滞留在S期,阻断向G2/M期发展,从而抑制肿瘤细胞增殖。相思子蛋白P2可明显诱导B16细胞凋亡。荧光显微镜检测结果显示相思子蛋白P2可显著降低B16细胞线粒体膜电位。相思子蛋白P2口服给药对小鼠B16移植性肿瘤生长有抑制作用,100μg/kg抑瘤率达53.75%,有明显剂量-效应依赖关系,且对胸腺和脾脏质量指数的影响较环磷酰胺小。结论:相思子蛋白P2于体内外均可显著抑制B16细胞的生长,此作用与阻止肿瘤细胞增殖周期,降低线粒体膜电位,诱导细胞凋亡有关。

Objective：To investigate the effects of abrin P2 on the inhibition of the proliferation of B16 melanoma cells,and to explore its mechanism.Methods In vitro：The effects of abrin P2 on B16 melanoma cells proliferation were evaluated by MTT assay.Flow cytometry analysis was applied to detect apoptosis,cell cycle distribution.JC-1 staining method was used to detect the effects of abrin P2 on the mitochondrial membrane potential of B16 melanoma cells.In vivo：Intragastrically administered abrin P2（50,75,100 μg/kg） to C57BL/6J mice for successive 10 days,B16 tumors xenografted in mice were used to evaluate the anti-tumor effects of abrin P2.Results：Cell proliferation was inhibited in B16 cells treated with abrin P2,and the value of IC50 was 4.6×10-3 μg/ml after 48 h.The inhibitory rate reached 95.45% when the concentration was 1μg/ml.Flow cytometry analysis showed,abrin P2 arrested the cell cycle at S phase and blocked cell grown to G2/M phase to inhibit tumor cell proliferation.Abrin P2 can significantly induced B16 cells apoptosis which showed a clear dose-dependent manner.The fluorescence microscope detection results showed that abrin P2 significantly reduced the mitochondrial membrane potential of B16 cells.In addition,the anti-tumor effects of abrin P2 were further confirmed by inhibition growth of B16 tumors xenografted in mice,with a tumor inhibition rate of 53.75% at the dose of 100 μg/kg.And the effects on thymus index and spleen index were lower compared to the cyclophosphamide group.Conclusions ：Abrin P2 can inhibit the proliferation of B16 melanoma cells in both vitro and vivo,and the effects may be related with the reduction of mitochondrial membrane potentia,the change of cell cycle distribution（S phase arrest） and the induction of cell apoptosis.