摘要
目的探讨丙戊酸钠对多发性骨髓瘤（MM）细胞株RPM18226和U266细胞自噬的影响。方法丙戊酸钠处理RPM18226和U266细胞，吖啶橙染色后采用荧光显微镜观察细胞自噬形态学变化，MTT法检测细胞增殖抑制的变化，流式细胞术检测细胞凋亡，实时定量PCR（RT-PCR）和WesternBlot法检测细胞自噬相关因子LC3、Beclin1的变化。结果荧光显微镜观察到RPM18226及U266细胞存在基础水平的自噬现象，丙戊酸钠作用后能够诱导细胞自噬增多；MTT法检测结果显示丙戊酸钠对细胞增殖抑制具有时间及浓度依赖性，作用24h后半数抑制浓度分别为（12．03±0．23）mmol／L和（10．16±0．37）mmol／L。8mmol／L丙戊酸钠作用24h后，RPM18226、U266细胞LC3mRNA表达水平（22．454-0．07、0．064-0．02）、Beclin1mRNA表达水平（283．094-17．3、1．53±0．01）与空白对照组（1．00：L0．00、1．00±0．00）比较，差异均有统计学意义（尸值均〈0．05）。随着丙戊酸钠浓度增加和作用时间延长，LC3、Beclin1蛋白表达水平逐渐增加，LC3I向LC3Ⅱ的转化率逐渐升高。结论RPM18226和U266细胞中存在基础水平的自噬现象，丙戊酸钠对MM细胞的自噬有激活作用，这可能是丙戊酸钠治疗MM的机制之一。
Objective To investigate the effects ofvalproic acid （VPA） on autophagy in multiple myeloma （MM） cell lines RPMI8226 and U266. Methods The method of dye acridine orange （AO） was used for observing morphological changes of autophagy under fluorescence microscope; The cell proliferation inhibition was measured by MTT assay; Cells apoptosis was evaluated by flow cytometry; Autophagy-related factors LC3, Beclinl expressions changes were detected by real-time quantitative PCR （Real-time PCR） and western blot assay. Results AO stainings as dispersively brownish red vesicles were observed both in the control and chloroquine groups, while a lot of brownish red acidic vesicles in clusters were seen in rapamycin and VPA groups. The growths of RPMI8226 and U266 cells were suppressed by VPA treatment in a dose- and time-dependent manner, after treatment with VPA for 24 h, the ICs0 were （12.03± 0.23） mmol/L for RPMI8226 cells and （10.16 ±0.37） mmol/L for U266 cells respectively; Poptotie ceils of RPMI8226 and U266 increased in a time-dependent manner after exposure to VPA. Real-time PCR and Western blot results of RPMI8226 and U266 cells showed that gradually increased LC3, Beclinl mRNA and protein expressions with LC3 I to LC3 II conversion rate after increasing the concentration of VPA and prolonging duration of action of VPA. Conclusions The results reveal disclosed the basal level of autophagy in MM cells, VPA as a autophagy activator may be one of its actions on the treatment of MM.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2016年第6期478-483,共6页
Chinese Journal of Hematology
基金
河北省自然科学基金（H2013406112）
