自噬对多发性骨髓瘤细胞增殖的影响

摘要

目的：探讨自噬（ autophagy）对正常培养多发性骨髓瘤（ Multiple myeloma，MM）细胞株增殖的影响。方法 MM细胞株U266在正常培养条件下分别加入雷帕霉素和3－甲基腺嘌呤（3-methyladenine，3-MA），以正常血清培养U266细胞及淋巴瘤Jurkat细胞株为对照组；分别检测细胞的增殖及凋亡水平；单丹磺酰尸胺（ monodansylcadaverine ，MDC）荧光染色法检测细胞自噬水平；逆转录PCR检测自噬相关基因mtor、Beclin1、Atg5在细胞内水平，蛋白质印迹法检测细胞内自噬相关蛋白轻链3I／Ⅱ（ LC3I／Ⅱ）含量。结果 U266细胞及Jurkat细胞内均存在自噬，但前者水平高于后者。雷帕霉素可诱导MM细胞发生自噬，3-MA对自噬有双重作用。雷帕霉素及3-MA下调细胞增殖水平。雷帕霉素及3-MA处理24 h可上调正常培养条件下U266细胞凋亡水平（1．7％±0．2％，19．1％±1．0％和16．8％±0．61％）。延长培养时间至72 h，雷帕霉素处理组凋亡水平无明显变化（16．9％±0．48％）。而3-MA处理组凋亡水平下降（9．0％±0．70％）。结论 MM细胞株内会存在一定水平的自噬，并通过实验证明其高于淋巴瘤Jurkat细胞株的自噬水平。在正常培养条件下加入了雷帕霉素及3-MA可抑制MM细胞的增殖，并可诱导细胞凋亡。%Objective To investigate the influence of autophagy on the proliferation of multiple myeloma ( MM) cells under normal cul-ture condition.Methods Multiple myeloma ( MM) cell line U266 was incubated with rapamycin or 3-MA respectively.Normally cul-tured Jurkat and U266 cell were used as control group.We determined the proliferation and apoptosis of cells respectively.Monodansyl-cadaverine( MDC) staining was employed to detect the autophagy.Reverse transcription PCR detected cells autophagy related genes mTOR,Beclin1,Atg5 level.Western blot was employed to detect the expression of protein LC3I /LCII.Results Autophagy level in U266 cells was higher than Jurkat cells.Rapamycin induced autophagy in U266 cell .3-MA showed inhibitory effect on autophagy for a short time.Rapamycin or 3-MA could induce cell apoptosis when incubated in normal culture condition for 24 hour (19.1%±1.0 %and 16.8%±0.61%,control group 1.7%±0.2%).Prolonged incubation time to 72 hours,the apoptosis level of rapamycintreatment group had no difference(16.9%±0.48%).But the levels of apoptosis of 3-MA group was decreased(9.0%±0.70%).Conclusion There was a basic level of autophagy in MM cell,and it was higher than those in the Jurkat cells.Rapamycin and 3-MA both inhibited the proliferation of MM cell and induced cell apoptosis.