摘要
目的探讨新型免疫抑制剂FTY720对多发性骨髓瘤细胞株U266细胞凋亡及自噬的影响，并探讨相关的作用机制。方法0、2．5、5．0、10．0及20．0μmol／LFTY720处理U266细胞24h后，应用CCK_8方法检测不同浓度FTY720对U266细胞生存率的影响；20．0μmol／LFTY720分别处理细胞0、2、6及24h，通过CCK-8方法检测FTY720作用不同时间对U266细胞生存率的影响。同时应用流式细胞术AnnexinV—FITC／PI染色检测相应的细胞凋亡率。不同浓度FTY720处理U266细胞后，应用Western blot法检测微管相关蛋白轻链3（LC3）B的表达，明确应用FTY720后是否引起细胞发生自噬。FTY720单用或联合应用自噬抑制剂Bafilomycin A1处理U266细胞24h，检测细胞生存率及凋亡率，并检测其对抗凋亡因子survivin表达的影响。结果应用FTY720后，U266细胞生存明显受抑制，可引起细胞发生凋亡，作用呈浓度及时间依赖性。LC3B-Ⅱ表达明显增加，呈浓度依赖性，表明FTY720可引起细胞发生自噬。应用自噬抑制剂Bafilomycin A1后，可解救FTY720引起的细胞生存抑制及凋亡，同时可解救FTY720引起的survivin表达下降。结论FTY720可引起U266细胞发生凋亡及自噬，自噬对凋亡具有促进作用。其机制可能为通过自噬在溶酶体降解了survivin等抗凋亡因子或其上游调控因子，从而促进细胞凋亡。
Objective To investigate the effects of FTY720, a new immunosuppresine agent, on ap- optosis and autophagy in multiple myeloma （MM） cell line U266 and to elarify its molecular mechanism. Methods U266 cells were treated with 0,2.5,5.0,10.0 and 20.0 μmol/L FTY720 for 24 hours, and the cell viability was assayed by CCK-8 method. Then U266 cells were treated with 20.0 μmol/L FFY720 for 0, 2,6 and 24 hours, the cell viability was tested. The apoptotic rates induced by different doses and time points of FTY720 were tested by flow cytometry separately. The expression of LC3B was detected by Western blot after U266 cells treated with different doses of FTY720 to see autophagy. U266 cells were treated with FTY720 ± Bafilomycin A1, an inhibitor of autophagy, for 24 hours, then the cell viability and apoptotic rates were tested. Meanwhile the expression of survivin, anti-apoptotic factors, were tested by Western blot. Results The cell viability and the apoptotic rates were inhibited significantly by FTY720 （ P 〈 0.05 ） in time- dependent and dose-dependentmanner. The expression of LC3B- 11 increased significantly in a dose-dependent manner, it indicated that the autophagy was induced by FTY720. Bafilomycin A1 could rescue the cell viability and apoptotic rates in U266 cells treated with FTY720, and it could also rescue the expression of survivin decreased by FTY720. Conclusions FFY720 can cause apoptosis and autophagy of U266 cells. The autoph- agy promote the apoptosis, which maybe due to the degradation of anti-apoptotic factors such as survivin or their upstream factors in lysosomes through autophagy.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2011年第10期664-667,共4页
Chinese Journal of Hematology
基金
辽宁省科技厅项目（2007225009-2）
