常春藤皂苷元影响内质网相关途径阻滞骨肉瘤细胞周期的实验研究

Effect of hederagenin on the cell cycle of osteosarcoma blocking the endoplasmic reticulum related pathway

目的:研究常春藤皂苷元对骨肉瘤细胞周期的影响及机制。方法:用0、5、10、20、40μg/ml的常春藤皂苷元处理骨肉瘤细胞,CCK-8法检测细胞增殖情况,细胞克隆实验测定细胞克隆形成能力,计算其半数抑制浓度。用半数抑制浓度的常春藤皂苷元处理骨肉瘤细胞,流式细胞术检测细胞周期和细胞凋亡,荧光探针法测定细胞内Ca^2+浓度。用Western blot法检测细胞中结合免疫球蛋白(BIP)、内质网氧化还原酶1-Lα(Erol-Lα)、蛋白质二硫键异构酶(PDI)、剪切型Caspase-12(Cleaved Caspase-12)、细胞周期蛋白D1(Cyclin D1)蛋白水平。结果:5、10、20、40μg/ml的常春藤皂苷元均可以抑制骨肉瘤细胞的增殖和克隆形成能力,与0μg/ml常春藤皂苷元作用组比较,差异有统计学意义(P<0.05)。半数抑制浓度的常春藤皂苷元处理后的骨肉瘤细胞G 1期比例升高,细胞凋亡率也升高,细胞内Ca^2+浓度升高,细胞中BIP、Erol-Lα、PDI、Cleaved Caspase-12蛋白水平升高,Cyclin D1蛋白水平降低,与0μg/ml常春藤皂苷元作用组比较,差异有统计学意义(P<0.05)。结论:常春藤皂苷元通过影响内质网相关途径将骨肉瘤细胞周期阻滞在G1期,诱导细胞凋亡发生。

Objective:To study the effect and its mechanism of hederagenin on the cell cycle of osteosarcoma.Methods:The osteosarcoma cells were treated with hederagenin of 0,5,10,20 and 40μg/ml.CCK-8 assay was used to detect cell proliferation.Cell cloning assay was used to determine the ability of cell clone formation.The half inhibitory concentration was calculated.The osteosarcoma cells were treated with a half inhibitory concentration of hederagenin.Cell cycle and apoptosis were detected by flow cytometry,The intracellular Ca^2+concentration was measured by fluorescence probe.The levels of BIP,Erol-L alpha,PDI,Cleaved Caspase-12 and protein in the cells were detected by Western blot.Results:The hederagenin of 5,10,20,40μg/ml could inhibit the proliferation and cloning of osteosarcoma cells.Compared with 0μg/ml hederagenin,the difference was statistically significant(P<0.05).The proportion of G 1 phase in the osteosarcoma cells treated by a half inhibitory concentration of hederagenin was increased.The rate of apoptosis was also increased,the concentration of Ca^2+in cells increased.The levels of BIP,Erol-Lα,PDI and Cleaved Caspase-12 were increased in the cells,the level of Cyclin D1 protein was reduced.Compared with 0μg/ml hederagenin,the difference was statistically significant(P<0.05).Conclusion:Hederagenin blocks cell cycle in G 1 phase,and induces apoptosis by the endoplasmic reticulum related pathway.