摘要目的 通过体外实验证实缺氧可诱导胶质瘤细胞的逆分化或维持胶质瘤干细胞的干性而导致化疗抵抗并探讨其机制.方法 实验采用U87、GL261胶质瘤细胞株,经磁珠分选获得CD133+ CD15+ Nestin+和CD133-CD15-Nestin-细胞.分选的细胞经常氧-缺氧培养后行免疫荧光实验和免疫印迹实验检测CD133、CD15、Nestin、缺氧诱导因子1α(HIF1α)的表达;进一步添加替莫唑胺(TMZ)后检测细胞凋亡情况及其半数抑制浓度(IC50).敲除HIF1α的细胞子缺氧培养后,以免疫印迹实验检测CD133、CD15、Nestin的表达;进一步添加TMZ后检测细胞凋亡情况及其IC50.结果 经TMZ处理的GL261 CD133+ CD15+ Nestin+细胞的凋亡率低于CD133-CD15-Nestin-细胞(P＜0.05),而IC50值高于后者(P＜0.05).免疫印迹实验结果表明,分选的U87细胞子缺氧培养后高表达CD133、CD15、Nestin以及HIF1α蛋白,而在常氧环境下几乎不表达上述蛋白(均P＜0.05).分选的U87、GL261细胞缺氧培养后,予TMZ处理后的细胞凋亡率较常氧条件下培养的细胞下降,IC50值升高(均P＜0.05);与未敲除HIF1α的细胞相比,敲除HIF1α的细胞中CD133、CD15、Nestin呈低表达(均P＜0.05),经TMZ处理后,其细胞凋亡率增加(均P＜0.05),细胞生存率下降(均P＜0.05).结论 缺氧可通过促进普通分化胶质瘤细胞的逆分化或维持胶质瘤干细胞的干性而导致化疗抵抗.

abstractsObjective To confirm that hypoxia can induce reverse differentiation of glioma cells or maintain the stemness of glioma cells,leading to chemotherapeutic resistance and to explore its mechanism by in vitro studies.Methods U87 and GL261 cells were considered as glioma cells line in this study.CD133 +CD15 + Nestin+ and CD133-CD15-Nestin-glioma cells were sorted through magnetic activated cell sorting and cultured in normoxia and hypoxia environments.CD133+ CD15 + Nestin+ and CD133-CD15-Nestin-glioma cells were cultured in hypoxia to detect the expressions of CD133,CD15,Nestin and hypoxia-inducible factor (HIF) 1α by Western blot and immunofluorescence assay.Cell apoptosis and half maximal inhibitory concentration (IC50) values were assessed after temozolomide (TMZ) treatment.HIF1α knocked-out glioma cells were cultured in hypoxia and the expressions of CD133,CD15 and Nestin were determined by Western blot.Besides,we compared cells apoptosis rate and IC50 values after TMZ treatment between HIF1α knockedout glioma cells and control group.Results GL261 CD133+ CD15+ Nestin+ cells showed lower cells apoptosis and higher IC50 value compared with Gl261 CD133-CD15-Nestin-cells after TMZ treatment (both P ＜ 0.05).The results of Western blot showed that the expression levels of CD133,CD15,Nestin and HIF1α in CD133 + CD15 + Nestin + and CD133-CD15-Nestin-U87 cells in hypoxia were higher than those in control group cultured in normoxia,which were barely expressed in the latter (all P ＜0.05).There were lower cells apoptosis and higher IC50 value after TMZ treatment in hypoxia in CD133 + CD15 + Nestin + and CD133-CD15-Nestin-U87 and GL261 glioma cells (all P ＜ 0.05).The expressions of CD133,CD15 and Nestin were decreased in HIF1α knocked-out glioma cells compared with those in cells without knocking out HIF1α.Higher cells apoptosis rate and lower IC50 value were reported for HIF1 α knocked-out glioma cells after TMZ treatment (all P ＜ 0.05).Conclusion Hypoxia could induce chemotherapy resistance by maintaining the stemness of glioma stem cells or promoting the reverse differentiation of normally differentiated glioma cells.