条件培养基抑制L-抗坏血酸钠介导的小鼠黑色素瘤细胞凋亡

目的探讨肿瘤细胞B16F10通过自分泌对抗由L-抗坏血酸钠(VitCNa)介导的凋亡作用。方法常规在75 cm~2培养瓶中培养小鼠黑色素瘤细胞B16F10,待细胞长至90%～95%融合时吸取原DMEM/高糖培养液,用PBS反复清洗后加入无血清的DMEM/高糖10ml在培养箱中培养6～8 h,收集培养液用2500 r/min离心5min后上清用0.22μm滤器过滤,-20℃保存备用,在6孔板中每孔接种1×10~6 B16F10,常规培养24h后,实验组每孔加入2ml条件培养液(含有10%胎牛血清),对照组用普通培养液,两组均用10 mmol/L VitC Na促凋亡,分别在3、6、24 h观察细胞凋亡情况,hoechst染核,收集细胞免疫荧光检测caspase3和TUNEL、RT-PCR检测Bcl2表达。进而分离出条件培养液中相对分子质量大于5 000和小于5 000成分,分别煮沸,用蛋白酶K灭活后对B16F10促凋亡。结果发现条件培养基能够抑制VitC Na介导的小鼠黑色素瘤细胞B16F10凋亡,抗凋亡成分小于5 000,且不能被煮沸和蛋白酶K灭活,提示其是小分子抗凋亡物质。结论 B16F10可以分泌出拮抗细胞凋亡的小分子物质。

Mouse melanoma cell line B16F10-derived conditioned medium inhibits sodium L-ascorbate-induced B16F10 cell apoptosis

Objective To investigate the effect of mouse melanoma cell line B16F10-derived conditioned medium on the apoptosis of B16F10 cells.Methods B16F10 cells were cultured in high-glucose DMEM in the presence of 10%fetal bovine serum,and upon cell confluence,the growth medium was replaced with serum-free high-glucose DMEM.After 8 h,the medium was collected and infiltrated to serve as the conditioned medium.B16F10 cells cultured in normal growth medium or the conditioned medium were exposed to 10 mmol/L sodium L-ascorbate,and the cell apoptosis was analyzed.The ingredients in the conditioned medium with relative molecular mass less or more than 5 000 were extracted to assess their effect on sodium L-ascorbate-induced cell apoptosis.Results The conditioned medium for B16F10 cells significantly inhibited cell apoptosis induced by sodium L-ascorbate,and the effective ingredients in the medium showed a relative molecular mass below 5 000.Conclusion Mouse melanoma cell line B16F10-derived conditioned medium can suppress sodium L-ascorbate-induced apoptosis of B16F10 cells,and the ingredients with relative molecular mass less than 5 000 are responsible for this effect.