3-甲基腺嘌呤诱发HeLa细胞自噬及抑制辐照细胞自噬的双重作用

Dual Effects of 3-Methyladenine on HeLa Cells Autophagy:Induction by the Action Alone and Inhibition on Autophagy Induced by Ionizing Radiation

目的了解PI3K抑制剂3-甲基腺嘌呤(3-MA)对电离辐射后细胞自噬的影响,初步探讨作用机制。方法细胞增殖-毒性试剂盒CCK-8检测不同浓度3-MA处理及60Coγ射线4Gy照射或联合3-MA作用的HeLa细胞细胞生长变化和毒性,AnnexinⅤ-FITC/PI双标记检测细胞凋亡,Western blot检测自噬相关蛋白SQSTM1/p62表达,Lipofectamine 2000介导转染GFP-LC3质粒,共聚焦显微镜检测自噬体形成。结果 3mmol/L以上浓度3-MA作用24h对HeLa细胞增殖有明显的抑制作用,作用更长时间产生致死毒性效应。无论是5mmol/L 3-MA单独作用还是单独4Gy照射,均能诱发HeLa细胞自噬。但3-MA与(射线辐照联合作用时,HeLa细胞自噬受到抑制,细胞凋亡显著增加。结论 PI3K抑制剂3-MA既诱发细胞自噬又能抑制电离辐射诱发细胞自噬,以在不同的条件下两种方式影响细胞存活。

Objective To investigate the cell suivival effect of phosphoinositide 3 - kinase（PI3K） inhibitors 3 - M ethyladenine （3 - MA） in response to ionizing radiation（IR） and the possible mechanism. Methods Firstly, HeLa cells were treated with 3 - MA in dif- ferent concentration,cell counting Kit -8 （CCK -8） assay were used to deteete the viability rate, Afterwards, HeLa cells were either treated with 5mmol/L 3 - MA or not treated, then irradiated with 60 Coγ - ray, cultivated for a certern time, cell counting kit - 8 （ CCK - 8） assay and annexin V - FITC/PI double staining were used respectively to detecte the viability rate and observe apoptosis rate in order to make a clear understanding about the cell proliferation toxic - effect of 3 - MA and IR. The GFP - LC3 puncta were examined under a confocal microscope after transfect GFP - LC3 by lipofectamin 2000. Western blot was performed to explore the expressions of autophagy - related proteins SQSTM1/p62. Results The proliferation inhibition and cytotoxicity of 3 - MA were demonstrated after the treatment for morn than 24h at the concentration 3mmol/L and higher. 5mmol/L 3 - MA action alone can induce HeLa ceils autophagy. However, 5mmol/L 3 - MA can inhibit the 4Gy γ- ray - induced HeLa cell autophagy, and promote the apoptosis. Conclusion The cell survival rate was impected in two ways, that were by inhibiting and inducing autophagy respectively after prolonged treated with the PI3 K inhibitors 3 - MA.