电离辐射对乳腺癌干细胞活性氧、凋亡和细胞周期的影响

doi:10.13481/j.1671-587x.20140602

吉林大学学报(医学版)

电离辐射对乳腺癌干细胞活性氧、凋亡和细胞周期的影响

方芳^1,2,赵雪莲¹,李丹利¹,白杨¹,龚守良¹,刘扬^1,3,王志成¹

(1. 吉林大学公共卫生学院卫生部放射生物学重点实验室，吉林长春 130021；2. 吉林大学公共卫生学院营养与食品卫生教研室，吉林长春 130021；3. 吉林大学公共卫生学院放射医学教学实验中心，吉林长春 130021)

收稿日期:2014-04-10 出版日期:2014-11-28 发布日期:2015-01-18
通讯作者: 王志成 E-mail:（Tel：0431-85619443，E-mail：zhicheng123a@126.com）
作者简介:方芳（1981-），女，吉林省白城市人，讲师，医学博士，主要从事低剂量电离辐射和乳腺癌干细胞的研究。
基金资助:
国家自然科学基金资助课题（30970681）；吉林大学白求恩青年科研基金资助课题（2013202017）；吉林大学创新训练项目资助课题（2013A72261）

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

FANG Fang ^1,2，ZHAO Xue-lian¹，LI Dan-li¹，BAI Yang¹，GONG Shou-liang¹，LIU Yang^1,3，WANG Zhi-cheng¹

（1. Key Laboratory of Radiobiology,Ministry of Health,School of Public Health,Jilin University,Changchun 130021,China;2. Department of Nutrition and Food Hygiene， School of Public Health，Jilin University, Changchun 130021，China；3. Radiation Medicine Teaching Experiment Center,School of Public Health,Jilin University,Changchun 130021,China)

Received:2014-04-10 Online:2014-11-28 Published:2015-01-18

摘要/Abstract

摘要：

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。

关键词: , 电离辐射；乳腺肿瘤；干细胞；悬浮培养；活性氧；细胞凋亡；细胞周期

Abstract:

Abstract: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-7 cells 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+8 Gy and MCF-7-S+8 Gy groups in the experiment.4-24 h after 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),AnnexinⅤ-FITC/PI and PI staining,respectively.Results The breast cancer stem cell microsphere accumulated hundreds of cells were obtained successfully at 7 d after 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-7 group 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-7 group at 4,8,12 and 24 h (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-7 group only at 8 h (P<0.05);the ROS levels (4,8,12 and 24 h) and percentage of apoptotic cells(12 h) were significantly increased in MCF-7+8 Gy group (P<0.05),and the percentages of apoptotic cells (4,8,12 and 24 h) in MCF-7-S +8 Gy group were significantly decreased (P<0.05 or P<0.01),but the ROS levels had no obvious change in MCF-7-S+8 Gy.At 12 h,as compared with MCF-7 group,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+8 Gy 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.

Key words: ionizing radiation;breast neoplasms, stem cells;suspension culture;reactive oxygen species；apoptosis；cell cycle

中图分类号:

R737.9

方芳1,2,赵雪莲1,李丹利1,白杨1,龚守良1,刘扬1,3,王志成1. 电离辐射对乳腺癌干细胞活性氧、凋亡和细胞周期的影响[J]. 吉林大学学报(医学版), 2014, 40(06): 1132-1137.

FANG Fang，ZHAO Xue-lian，LI Dan-li，BAI Yang，GONG Shou-liang，LIU Yang，WANG Zhi-cheng. Effects of ionizing radiation on ROS,apoptosis and cell cycle in breast cancer stem cells[J]. Journal of Jilin University Medicine Edition, 2014, 40(06): 1132-1137.

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电离辐射对乳腺癌干细胞活性氧、凋亡和细胞周期的影响

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

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