电离辐射对乳腺癌干细胞活性氧、凋亡和细胞周期的影响

目的：研究电离辐射对乳腺癌细胞和乳腺癌干细胞活性氧(ROS)、凋亡和周期分布的影响，探讨乳腺癌干细胞辐射抵抗产生的机制。方法：采用含多种生长因子的无血清培养基悬浮培养人乳腺癌MCF-7细胞系，分为乳腺癌细胞MCF-7组、乳腺癌干细胞MCF-7-S组、MCF-7+8 Gy组和MCF-7-S+8 Gy组。8 Gy电离辐射后4~24 h，分别采用2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)、AnnexinⅤ-FITC/PI和PI染色，流式细胞术检测细胞中ROS水平、凋亡细胞百分比和各细胞周期时相细胞百分比。结果：含多种生长因子无血清悬浮培养7 d后，形成数百个细胞聚集而成的乳腺癌干细胞微球；流式细胞术检测，CD4++CD24－表型的乳腺癌干细胞高达75.20%。随着时间的延长，MCF-7组和MCF-7-S组细胞中ROS水平和细胞凋亡均呈增加的趋势，在12和24 h　达到较大值；在4、8、12和24 h时，MCF-7-S组细胞中ROS水平均低于MCF-7组（P<0.05或P<0.01），MCF-7-S组凋亡细胞百分比只在8 h显著高于MCF-7组细胞（P<0.05）；MCF-7+8 Gy组细胞中ROS水平（4、8、12和24 h)和凋亡细胞百分比（12 h）显著增加（P<0.05），MCF-7-S+8 Gy组凋亡细胞百分比（4、8、12和24 h）显著降低（P<0.05或P<0.01），MCF-7-S+8 Gy组细胞中ROS水平无明显变化。在12 h时，与MCF-7组比较，MCF-7-S组细胞G0/G1期和G2/M期细胞百分比显著降低（P<0.05），S期细胞百分比显著增加（P<0.05）；MCF-7+8 Gy组细胞G2/M期细胞百分比显著增加（P<0.05），而MCF-7-S+8 Gy组无明显变化。结论：电离辐射导致MCF-7中ROS水平和凋亡增加，G2/M期阻滞，而对MCF-7-S则无明显影响，提示MCF-7-S辐射抵抗可能与ROS水平、细胞凋亡和G2/M期阻滞有关。

Effects of ionizing radiation on ROS,apoptosis and cell cycle in breast cancer stem cells

Objective To explore the mechanism of radio-resistance of breast cancer stem cells by investigating the effects of ionzing radiation on the reactive oxygen species(ROS),apoptosis and cycle distribution.Methods The breast cancer MCF-7cells were suspension cultured in serum-free medium containing a variety of growth factors.There were MCF-7(breast cancer cells),MCF-7-S(breast cancer stem cells),MCF-7+8Gy and MCF-7-S+8Gy groups in the experiment.4-24 hafter 8 Gy irradiation,the ROS levels,percentages of apoptotic cells and percentages of the cells at each cycle phage were measured by FCM with 2′,7′-dichlorodihydrofluorescin diacetate(DCFH-DA), Annexin Ⅴ-FITC/PI and PI staining,respectively.Results The breast cancer stem cell microsphere accumulated hundreds of cells were obtained successfully at 7dafter suspension culture with serum-free medium containing a variety of growth factors;the FCM results showed that CD44+CD24-phenotype breast stem cells were up to 75.20%.With the time prolongation,the ROS levels and apoptosis in MCF-7group and MCF-7-S group showed increasing trendency,and reached for the maximum values at 12 and 24 h;the ROS levels in MCF-7-S group were significantly lower than those in MCF-7group at 4,8,12 and 24h(P<0.05 or P<0.01),and the percentage of apoptotic cells in MCF-7-S group was significantly higher than that in MCF-7group only at8h(P<0.05);the ROS levels(4,8,12 and 24h)and percentage of apoptotic cells(12h)were significantly increased in MCF-7+8 Gy group(P<0.05),and the percentages of apoptotic cells(4,8,12 and 24h)in MCF-7-S +8Gy group were significantly decreased(P<0.05 or P<0.01),but the ROS levels had no obvious change in MCF-7-S+8Gy.At 12 h,as compared with MCF-7group,the percentages of the cells at G0/G1 phase and G2/M phase in MCF-7-S group were significantly decreased(P<0.05),and the percentage of the cells at S phase was significantly increased(P<0.05);the percentage of the cells at G2/M phase in MCF-7+8Gy group was significantly increased(P <0.05),but there were no significant changes in MCF-7-S+8 Gy group.Conclusion Ionizing irradiation can cause the increasing of ROS level and apoptosis and G2/M phase arrest in breast cancer cells,but has no obvious effects on the breast cancer stem cells;it indicates that radio-resistance might be related to ROS level,apoptosis and G2/M phase arrest.