喉癌干细胞在低氧介导的喉癌放疗抵抗中的作用机制

Cancer stem cells play an important role in resistance of laryngeal squamous cancer to irradiation mediated by hypoxia

目的 体外观察低氧和常氧培养条件下喉癌CD133＋细胞放疗后生长抑制率、克隆形成率的变化和DNA依赖蛋白激酶催化亚单位（DNA dependent protein kinase catalytic subunit, DNA-PKcs）、共济失调毛细血管扩张症突变基因（ataxiatelangiectasia mutate,ATM）、Survivin、P53的表达,探讨肿瘤干细胞在低氧介导的喉癌放疗抵抗中的作用机制.方法 体外培养人喉癌Hep-2细胞,用流式细胞仪分选出CD133＋细胞,并分别在低氧和常氧条件下行无血清培养,流式细胞仪检测缺氧诱导因子-1α的表达.低氧组和常氧组分别用直线加速器给予0、5、10、15、20Gy的照射,24h后行四甲基偶氮唑蓝（MTT）检测；给予10 Gy照射后12、24、36、48 h行MTT检测.行软琼脂克隆形成实验,并给予10 Gy照射,14 d后观察低氧和常氧组克隆形成率.两组细胞分别给予10 Gy照射,24h后用流式细胞仪分别检测DNA-PKcs、ATM、Survivin、P53的表达.结果 流式细胞仪分选后获得CD133＋细胞纯度是92.8％,CD133＋细胞常氧组各个剂量和时间点的生长抑制率均高于低氧组；在低氧和常氧条件下,CD133＋细胞放疗后生长抑制率随放疗剂量的增加而增加,而10 Gy放疗后各个时间点细胞生长抑制率变化不大,24h时差异最大,具有统计学意义（t=6.12,P均＜0.05）.软琼脂克隆形成实验显示CD133＋细胞克隆形成率明显高于CD133-的对照组（P＜0.01）,CD133＋细胞低氧组的克隆形成率明显高于常氧组（P＜0.05）.放疗前后低氧组克隆形成率变化不明显,而常氧组放疗后克隆形成率较放疗前低（P＜0.05）.CD133＋细胞的DNA-PKcs、ATM、Survivin和P53的表达均高于CD133-细胞（P＜0.01）.放疗后两组细胞各种蛋白表达均高于放疗前（P＜0.01）,低氧组放疗后DNA-PKcs、Survivin表达均较常氧组高（P＜0.05）,而ATM、P53表达差异无统计学意义（P＞0.05）.结论 喉癌干细胞在低氧介导的喉癌放疗抵抗中有重要作用,多种通路参与了喉癌干细胞放射损伤的修复.

Objective To study whether laryngeal cancer stem cells in hypoxia have the characteristic of resistance to irradiation and underlying mechanism.Methods CD133 ＋ cells were separated from Hep-2 cells with flow cytometry（FCM）and the purity was 92.8%.The separated CD133 ＋ cells were cultured in serum-free medium in hypoxia in normoxia enviroment respetively,and hypoxiainducible factor 1 alpha（HIF-1α）expression was detected by FCM.The cells were exposed respectively to X-rays emitted by linear accelerator with a dose of 0,5,10,15 or 20 Gy for 24 hours,with additional time points of 12,36,and 48 hours for the cells exposed to 10 Gy.Then the growth inhibition ratios of ceils in hypoxia and normoxia groups were detected with MTT assay at different time points.Soft agar colony formation assay was used to dectect colony formation ratios of cells in hypoxia and normoxia groups.DNA dependent protein kinase catalytic subunit（DNA-PKcs）,ataxiatelangiectasia mutate（ATM）,Survivin and P53 were detected by FCM.Results Growth inhibition ratio of CD133 ＋ cells in hypoxia group was lower than that in normoxia group（P 〈0.05）.Colony formation ratio of CD133 ＋ cells was higher than that of CD133-cells（P 〈 0.01）and the ratio of CDi33 ＋ cells in hypoxia group was higher than that in nonnoxia group（P 〈0.05）.The ratio of hypoxia group was not affected by irradiation,while the ratio of normoxia group decreased significantly after irradiation（P 〈 0.05）.The expressions of DNA-PKcs,ATM,Survivin and P53 in CD133＋ cells were higher than those in CD133-cells respectively（P 〈0.01）.In CD133 ＋ cells with radiation,the expressions of DNA-PKcs and Survivin of hypoxia group were higher those of normoxia group（P 〈 0.05）,but no difference in the expression of ATM or P53 between the two groups.Conclusions Laryngeal cancer stem cells play an important role in radioresistance mediated by hypoxia.