摘要
目的探讨诱导自噬对多发性骨髓瘤（MM）细胞株增殖的影响。方法在无血清培养条件下诱导MM细胞株U266细胞自噬并分别加入雷帕霉素和3-甲基腺嘌呤（3-MA），观察其细胞增殖，以正常培养条件培养U266细胞、淋巴瘤细胞株Jurkat及无血清培养Jurkat为对照组。采用CCK8法检测细胞的增殖；用流式细胞术检测细胞凋亡；单丹（磺）酰戊二胺（MDC）法检测细胞自噬泡数量；RT—PCR法检测自噬基因mtor、Beclinl表达；Westernblot方法分析自噬标志蛋白-微管相关蛋白轻链3Ⅰ／Ⅱ（LC3I／II）及溶酶体相关膜蛋白1（LAMP1）含量的变化。结果正常培养条件下U266细胞有基础水平的自噬，无血清培养可诱导U266细胞上调自噬水平；雷帕霉素促进无血清培养条件下U266细胞自噬并刺激其增殖能力，至培养96h无血清培养及雷帕霉素处理U266细胞内自噬水平下降；3-MA具有上调U266细胞自噬和促进增殖作用，但仅维持24h；正常培养条件下雷帕霉素及3-MA可显著抑制U266细胞增殖；无血清培养24h后细胞凋亡率[（17．90±1．46）％]高于正常培养细胞[（1．33±0．09）％]（P〈0．01）；加入雷帕霉素及3-MA可使血清饥饿诱导的细胞凋亡率分别降至（6．23±0．12）％和（6．97±0．03）％（P〈0．01）；培养至72h，各处理组细胞凋亡率趋于一致[（30．37±0．27）％、（30．13±1．93）％和（28．57±2．83）％]（P〉0．05）；去除雷帕霉素及3-MA后U266细胞凋亡率减低至18．7％和12．6％。结论MM细胞内存在高于淋巴瘤Jurkat细胞株基础水平的自噬；在常规培养条件下雷帕霉素及3-MA均可下调MM细胞增殖水平；血清饥饿条件下U266细胞可通过上调自噬水平维持生存和增殖；雷帕霉素诱导血清饥饿条件下U266细胞自噬，但是具有白限性；3-MA对U266细胞自噬具有双重作用。
Objective To investigate the influence of autophagy on the survival and proliferation of multiple myeloma （MM） cells. Methods Multiple myeloma （MM） cell line U266 cell autophagy was induced by serum-free culture condition, and adding rapamycin or 3-MA respectively. The cells proliferation was observed. U266 cells, lymphoma cell Jurket under normal culture condition, and serum-free cultured Jur-ket cell were used as control group. The proliferation and apoptosis of ceils were determined by CCK8 and flow cytometry, respectively. MDC staining were employed to detect the autophagy. The mRNA expression of mtor and Beclinl gene of U266 cells were assayed by RT-PCR. Protein LC3Ⅰ/ LCⅡ and LAMP1 was analyzed by western blot. Results There was low level of autophagy in U266 cells , sera starvation increased the level of autophagy . Rapamycin upregulated autophagy of the U266 ceils and stimulated their proliferation. But the autuphagy level of sera starvation and rapamycin group declined when culture for 96h. 3-MA had the same effects on U266 cells, although it was on 24 h. But rapamycin and 3-MA could inhibit cell proliferation under normal culture condition. Compared with normal culture condition , apoptosis of U266 ceils increased signifi- cantly after 24h incubation in medium without sera [ （ 1.33±0.09） % and （ 17.90±1.46） %, respectively ] （P 〈 0.01 ）. Rapamycin and 3-MA could inhibit the serum-free induced apoptosis [ （6.23±0.12）% and （6.97±0.03 ） %, respectively ] （ P 〈 0.01 ）, but cell apoptosis was at the same level after 72 hour incubation [（ 30.37±0.27 ） % , （ 30.13±1.93 ） % and （ 28.57±2.83 ） % , respectively （ P 〉 0.05 ）. However, apoptosis of 15266 cells decreased to 18.7% and 12.6% after removal of rapamycin and 3-MA. Conclusion There is basical level of autophagy in MM cells which is higher than those in the Jurkat cells. Both Rapamycin and 3-MA can inhibit the cells proliferation under normal culture condition. Up-regulated autophagy promotes survival and proliferation of MM cells under sera deletion. Rapamycin strengthens this effect with limited duration. 3-MA has dual effects on cell autophagy.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2012年第11期932-937,共6页
Chinese Journal of Hematology
基金
国家自然科学基金（81071934）
江苏省优势学科建设经费资助
