微环境乏氧通过肿瘤干细胞途径对脑胶质瘤细胞放射敏感性的影响

Effect of microenviroment hypoxiaon glioma cells radiosensitivity through cancer stem cell pathway

目的探讨微环境乏氧是否能通过肿瘤干细胞途径引起脑胶质瘤放射敏感性的改变以及可能的相关机制。方法选择脑胶质瘤SHG44和U251细胞株分别在常氧（20％O2）、乏氧（1％O2）12和24h的条件下培养后，应用流式细胞仪检测CD133表达阳性细胞的比例，细胞克隆形成实验绘制细胞存活曲线以观察其放射敏感性，采用Western blotting方法测定基因HIF-1α及其下游基因Notch1蛋白的表达水平。结果与常氧培养条件比，SHG44和U251细胞乏氧12和24h后CD133阳性表达比例明显升高；SF2（2Gy照射时的存活分数）均升高。在乏氧12和24h培养时，SHG44细胞株的氧增强比的值分别为1．54和1．38，而U251细胞株则分别为1．44和1．23，提示在乏氧培养时细胞的放射敏感性下降；与常氧培养条件比，乏氧时HIF-1α和Notch1的蛋白表达水平均明显升高。结论微环境乏氧能通过提高肿瘤干细胞的比例而降低脑胶质瘤细胞的放射敏感性，其可能的作用信号途径为HIF-1α-Notch1。

Objective To investigate the effect of microenviromeut hypoxia on glioma cells radiosensitivity through cancer stem pathway, and to explore the related mechanism. Methods Glioma cell lines SHG44 and U251 were cultured in normoxia（20% O2 ） or continuous hypoxia（ 1% O2 ）for 12 and 24 h. The fraction of glioma cells with positive expression of CD133 was assayed by flow cytometry. The radiosensitivity of glioma ceils was determined by clonogenic cell assay. Western blotting was used to investigate the expressions of HIF-1α and its downstream gene Notch 1. Results The fraction of glioma cells with positive expression of CD133 was higher after hypoxic culture for 12 and 24 h than that of the corresponding cells cultured in normoxia. Compared to the cells cultured in normoxia, SF2 （survival fraction at 2 Gy） were enhanced significantly in SHG44 and U251 cells cultured in hypoxia for 12 and 24 h. The OER （oxygen-enhancement ratio） of SHG44 cells in hypoxia for 12 and 24 h was 1.54 and 1.38, respectively. The OER of U251 cells was 1.44 and 1.23, respectively. The radiosensitivity of these two cell line was decreased in hypoxia. The protein expressions of HIF-1α and Notchl genes were elevated more significantly for cells cultured in hypoxia for 12 and 24 h than for those in normoxia. Conclusions Mieroenviroment hypoxia could increase the radioresistance of glioma cells through enrichment of cancer stem celia, and HIF-1α - Notchl signal pathway may play an important role in this process.