中文摘要：

目的 以结肠癌细胞HCT116为研究对象,评价DNA依赖蛋白激酶催化亚基（DNA-PKcs）抑制剂NU7026对癌干细胞的放射增敏作用及其机制。方法 以CD133＋/CD44＋为标志物,流式细胞术检测癌干细胞亚群。将HCT116细胞分为对照组、单纯药物（20 μmol/L NU7026）组、单纯照射（2 Gy γ射线）组和药物＋照射（20 μmol/L NU7026联合2 Gy γ射线）组,照射前2 h加入NU7026。细胞集落形成实验检测HCT116细胞增殖,流式细胞术检测细胞周期和凋亡, γ-H2AX foci的免疫荧光激光共聚焦分析DNA双链断裂损伤修复。结果 体外培养HCT116细胞中CD133＋/CD44＋癌干细胞亚群比例高达（88.14±0.47）%,HCT116细胞低密度接种无血清培养基中集落形成率为（84.75±1.35）%。与单纯照射组比较,药物＋照射组细胞存活率显著降低（t=7.22,P〈0.01）。受照后48 h,药物＋照射组的癌干细胞亚群比例较单纯照射组显著降低（t=9.55,P〈0.01）。受照后24 h,NU7026明显增加G2/M期阻滞（t=7.67,P〈0.01）,48 h细胞早期凋亡发生率也明显增加（t=8.24,P〈0.05）。药物＋照射组在照后2、4、8和24 h DNA双链断裂（γ-H2AX foci）残留比单纯照射组明显增多（t=19.58、11.95、7.01和9.45,P〈0.01）。结论 NU7026对癌干细胞为优势亚群的结肠癌HCT116细胞具有明显的放射增敏作用,显著增加对癌干细胞的杀伤效应,增敏机制包括抑制DNA修复,诱发不可逆G2/M期阻滞和增加细胞凋亡。

英文摘要：

Objective To evaluate the radiosensitization and mechanism of DNA-dependent protein kinase catalytic subunit inhibitor NU7026 on HCT116 colorectal cancer stem cells.Methods The flow cytometry was used to determine the sub-population of cancer stem cells with the markers CD133/CD44. The cells were divided into four groups： control group, 20 μmol/L NU7926 group, 2 Gy irradiation group, and 20 μmol/L NU7026 combined with 2 Gy irradiation group. Cell proliferation and survival were evaluated by colony-formation experiment. Flow cytometry was used to analyze the cell cycle distribution and apoptosis induction. γ-H2AX foci were detected with immune-fluorescence staining analysis by a laser confocal microscopy for investiating the DNA double-strand break repair. Results The flow cytometric data of CD133＋/CD44＋ positive cells indicated that the sub-population of cancer stem cell （CSC） took the ratio of （88.14±0.47）% of the cultured HCT116 cells. The colony-formation efficiency of HCT116 cells was （84.75±1.35）% in serum-free mediums in vitro culture. Compared to 2 Gy irradiation alone group, the NU7026 combined with 2 Gy irradiation group had a lower cell colony-forming ratio （t=7.21, P〈0.01） and a lower survival ratio （t=7.22, P〈0.01）. The proportion of CSCs sub-population increased at 48 h post-2 Gy irradiation, suggesting that HCT116 CSCs was more resistant to ionizing radiation. Importantly, NU7026 largely decreased the proportion of CSCs sub-population in 2 Gy-irradiated cells. The difference of CSC proportion between 2 Gy irradiation alone group and 2 Gy combined with NU7026 treatment group was statistically significant （t=9.55, P〈0.01）. In addition, the group of NU7026 combined with 2 Gy irradiation had a higher ratio of G2/M arrest 24 h post-irradiation （t=7.67, P〈 0.01） and an increased induction of cell early apoptosis （t=8.24, P〈 0.05）. 48 h post irradiation as compared to 2 Gy irradiation alone group. NU7026 treatment significantly inhibited the cellular cap