摘要
目的观察胶质瘤干祖细胞(GSPCs)诱导分化前后自噬活性的变化,探讨GSPCs分化与自噬的相关性。方法于干细胞培养基和诱导分化培养基培养GSPCs,分别采用微管相关蛋白轻链3(LC3)免疫荧光染色、透射电镜及Western blot检测GSPCs分化前后自噬活性的变化。GSPCs诱导分化时,加入3-甲基腺嘌呤(3-MA)自噬抑制剂,观察其对GSPCs分化的影响,并采用自噬激活剂雷帕霉素(RPM)行进一步检测。结果 GSPCs分化前,自噬活性低;诱导其分化后,自噬活性增高。加入自噬抑制剂3-MA可抑制GSPCs贴壁分化;Western blot检测示,加入3-MA的GSPCs中胶质纤维酸性蛋白(GFAP)、微管相关蛋白-2(MAP-2)及beclin-1表达量显著低于未加3-MA的GSPCs。GSPCs在含血清条件下培养时,适当浓度的RPM可促进其贴壁分化。结论 GSPCs分化与其自噬活性密切相关。增强GSPCs自噬活性可促进分化,抑制其自噬活性可抑制分化。
Objective To explore the correlation between the differentiation and autophagy in GSPCs （glioma stem/progenitor cells） by observing the change of autophagy activity before and after GSPCs induction. Methods GSPCs were respectively cultured in stem cell medium,induction and differentiation medium. Before and after GSPCs induction,the changes of autophagy activity was detected using microtuhule-associated protein light chain-3 （IL-3） immunofluorescent staining,transmission electron microscope and Western blot methods. Autophagy inhibitor 3-MA （3-methyladenine） was added to observe its effect on the differentiation of GSPCs at the time of induction and differentiation,and RPM（rapamycin）, an autophagy promoter, was also used to perform further detection. Results Before differentiation, the autophagy activity of GSPCs was low while it increased markedly after inducing the differentiation of GSPCs. Additional autophagy inhibitor 3-MA could inhibit GSPCs differentiation. Western blot assay displayed the expression of GFAp （glial fibrillary acidic protein）, MAP-2 （microtubule-associated protein-2） and becliwl in GSPCs with 3-MA was dramatically higher than those without 3-MA. Appropriate concentration of RPM could accelerate the differentiation of GSPCs cultured in serum. Conclusion The differentiation of GSPCs is closely related to its autophagy activity. Enhancing the autophagy activity of GSPCs can promote its differentiation, while inhibiting it can inhibit the differentiation.
出处
《成都医学院学报》
CAS
2015年第4期404-409,共6页
Journal of Chengdu Medical College
基金
浦东新区卫生系统优秀青年医学人才培养计划(No:PWRq2012-39)
