目的：研究AKT2基因在U251细胞株中对替莫唑胺化疗耐药中的作用机制。方法体外将慢病毒介导的AKT2-shRNA表达载体转染胶质瘤U251细胞。应用细胞计数试剂盒（CCK8）法检测RNA干扰AKT2后U251细胞对替莫唑胺敏感性的变化情况。流式细胞仪测定沉默AKT2基因表达后各组细胞凋亡的改变情况。 AKT2-shRNA干扰胶质母细胞瘤细胞株U251的AKT2表达，进而采用Western blot实验方法，来进一步分析和评价AKT2与MDR-1、MRP-1、MGMT、bcl-2、caspase-3、P53等相关蛋白表达情况和相互调控关系。结果 CCK8法检测结果计算替莫唑胺对U251细胞的半数细胞抑制浓度（ IC50）从 U251空白对照组的（39．72±2．41）μg／ml、阴性对照组的（39．43±2．24）μg／ml降到（27．23±1．93）μg／ml，AKT2干扰组U251细胞对替莫唑胺药物的IC50值显著降低。流式细胞仪检测凋亡实验显示，与空白对照的U251细胞[调亡细胞（16．95±1．32）％]和转染阴性对照的U251胶质瘤细胞[调亡细胞（17．93±2．29）％]相比，转染 AKT2 shRNA的U251细胞中早期、晚期调亡细胞明显增加，总调亡细胞达（38．16±4．83）％，干扰组凋亡水平有显著性增强（P＜0．05）。 AKT2-shRNA干扰U251后其凋亡明显增加，Real-time PCR和蛋白印迹实验结果提示bcl-2、survivin、MGMT明显下调，而caspase-3、PTEN、P53、beclin-1明显上调。结论 AKT2可能参与调控bcl-2、caspase-3、P53、survivin、beclin1等凋亡自噬相关蛋白及DNA损伤修复蛋白MGMT的表达，从而介导胶质母细胞瘤细胞株U251对替莫唑胺化疗抵抗。%Objective To explore the effects of AKT2 expression in U251 glioma cells on the sensitivity towards to temozolomide ( TMZ).Methods The lentivirus vector of AKT2 shRNA was constructed and transfected into U251 cells.The changes of TMZ sensitivity after shRNA were examined by the CCK8 assay.Apoptosis of cells of each group was detected by flow cytometry cell technology.further analysis and evaluation of the AKT2 and the MDR-1, MRP-1, MGMT, and apoptosis related protein expression regulatory interaction used by Real-time PCR and Western Blot . Results the IC50 of U251 cell decreased from the blank control group (39.72 ±2.41)μg/ml, negative control group (39.43 ±2.24)μg/ml to (27.23 ±1.93) μg/ml,sensitivity of U251 cell to TMZ decreased significantly .Western blot results showed Bcl-2, survivin, MGMT was down regulated,and Caspase-3,PTEN,P53,beclin-1 increased.Conclusion AKT2 may be involved in the regulation of expression of apoptosis and autophagy related protein Bcl-2, Caspase-3, P53, survivin, Beclin1, and DNA damage repair protein MGMT , which mediates U251 chemotherapy resistance to TMZ.
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