电离辐射对Beclin1过表达和低表达MCF-7细胞模型细胞自噬和凋亡的影响及其调控机制

doi:10.13481/j.1671-587x.20150608

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (06): 1139-1143.doi: 10.13481/j.1671-587x.20150608

电离辐射对Beclin1过表达和低表达MCF-7细胞模型细胞自噬和凋亡的影响及其调控机制

张晶¹, 赵大力², 谢忠伟², 刘彦军³, 李志君¹, 李岩¹, 龚守良⁴, 齐亚莉¹

1. 北华大学公共卫生学院流行病学教研室, 吉林吉林 132011;
2. 吉林(市)出入境检验检疫局, 吉林吉林 132012;
3. 吉林省长春市人民医院肿瘤治疗科, 吉林长春 130051;
4. 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

收稿日期:2015-09-09 发布日期:2016-01-11
通讯作者: 齐亚莉,副教授,硕士研究生导师(Tel:0432-64608343,E-mail:374494617@qq.com) E-mail:374494617@qq.com
作者简介:张晶(1973-),女,吉林省九台市人,讲师,医学硕士,主要从事细胞毒理学方面的研究。
基金资助:
国家自然科学基金资助课题(30970681);吉林省教育厅"十二五"科学技术研究项目资助课题(2014-192)

Effects of ionizing radiation on autophagy and apoptosis in MCF-7 cells with Beclin 1 over-and low-expressions and their regulating mechanisms

ZHANG Jing¹, ZHAO Dali², XIE Zhongwei², LIU Yanjun³, LI Zhijun¹, LI Yan¹, GONG Shouliang⁴, QI Yali¹

1. Department of Epidemiology, School of Public Health, Beihua University, Jilin 132001, China;
2. Jilin Province (City) Entry and Exit Inspection and Quarantine Bureau, Jilin 132012, China;
3. Department of Radiotherapy, People's Hospital in Changchun City, Jilin Province, Changchun 130051, China;
4. Key Laboratory of Radiobiology, Ministry of Health, School of Public Health, Jilin University, Changchun 130021, China

Received:2015-09-09 Published:2016-01-11

摘要/Abstract

摘要：

目的:建立Beclin 1过表达和低表达MCF-7细胞模型,检测4 Gy照射后细胞自噬和凋亡的变化,探讨Beclin 1的分子调控作用机制。方法:实验分为MCF-7组、MCF-7+4Gy组、MCF-7-Beclin1+4Gy组和MCF-7-Belin1 RNAi+4Gy组。利用分子生物学方法构建Beclin 1过表达载体pcDNA3.1-Beclin 1,并建立Beclin 1过表达和低表达细胞模型。细胞经4 Gy照射后,采用MDC染色荧光显微镜观察自噬细胞百分比,AnnexinⅤ-FITC和PI染色流式细胞术检测凋亡细胞百分比,Western blotting法检测Beclin 1、P53、Bcl-2和Bax蛋白表达。结果:与MCF-7组比较,MCF-7+4 Gy组、MCF-7-Beclin 1+4 Gy组和MCF-7-Beclin 1 RNAi+4 Gy组自噬和凋亡细胞百分比均明显升高(P<0.05 或 P<0.01),以MCF-7-Beclin 1+4 Gy组升高最明显,且高于MCF-7+4 Gy组 (P<0.05);各组坏死细胞百分比无明显差异。4 Gy照射后,MCF-7组和MCF-7-Beclin 1组细胞中Beclin 1、P53和Bax蛋白表达水平均升高,Bcl-2蛋白表达水平降低,且以MCF-7-Beclin 1组最为明显。结论:成功建立Beclin 1过表达和低表达MCF-7细胞模型,电离辐射能够诱导其细胞自噬和凋亡,且对Beclin 1过表达细胞作用更明显。Beclin1通过激活P53,进而抑制Bcl-2和激活Bax,从而形成以P53为中心的自噬与凋亡分子调控机制。

关键词: 自噬, 细胞凋亡, 电离辐射, Beclin 1, P53

Abstract:

Objective To establish the MCF-7 cell models of Beclin 1 over-and low-expressions, and to detect the autophagic and apoptotic changes after 4 Gy irradiation, and to explore their molecular regulation mechanisms. Methods MCF-7,MCF-7+4Gy,MCF-7-Beclin 1+4Gy and MCF-7-Belcin 1 RNAi+4Gy groups were set up. Molecular biology method was used to construct Beclin 1 over-expression vector pcDNA3.1-Beclin 1, and to estabilish the Beclin 1 over-and low-expression cell models. After the cells were irradiated with 4 Gy, the autopahgic cell percentages were measured by fluorescence microscope with MDC staining, the apoptotic cell percentages were measured by FCM with AnnexinⅤ-FITC and PI staining, and the expressions of Beclin1, P53, Bcl-2 and Bax proteins were measured by Western blotting method. Results Compared with MCF-7 group, the autophagic and apoptotic cell percentages in MCF-7+4 Gy, MCF-7 Beclin 1+4 Gy and MCF-7-Beclin 1 RNAi+4 Gy groups were significantly increased (P<0.05 or P<0.001 ), especially in MCF-7 Beclin 1+4 Gy group which was significantly higher than those in MCF-7+4 Gy (P<0.05); while there was significant difference in the necrotic cell percentages between various groups. After 4 Gy irradiation, compared with MCF-7 group, the expression levels of Beclin 1, P53 and Bax proteins in MCF-7+4 Gy and MCF-7-Beclin 1+4 Gy groups were increased, but the expression levels of Bcl-2 protein were decreased, especially in MCF-7-Beclin 1+4 Gy group. Conclusion The MCF-7 cell models of Beclin 1 over-and low-expressions are successfully established, and ionizing radiation could induce the autophagy and apoptosis of MCF-7 cells, which is more obvious in Beclin 1 over-expression MCF-7 cells. Beclin 1 can activate P53, inhibit Bcl-2 and activate Bax,which forms the regulation of autophagy and apoptosis by P53.

Key words: autophagy, aopotosis, ionizing radiation, Beclin 1, P53

中图分类号:

R818

张晶, 赵大力, 谢忠伟, 刘彦军, 李志君, 李岩, 龚守良, 齐亚莉. 电离辐射对Beclin1过表达和低表达MCF-7细胞模型细胞自噬和凋亡的影响及其调控机制[J]. 吉林大学学报(医学版), 2015, 41(06): 1139-1143.

ZHANG Jing, ZHAO Dali, XIE Zhongwei, LIU Yanjun, LI Zhijun, LI Yan, GONG Shouliang, QI Yali. Effects of ionizing radiation on autophagy and apoptosis in MCF-7 cells with Beclin 1 over-and low-expressions and their regulating mechanisms[J]. Journal of Jilin University Medicine Edition, 2015, 41(06): 1139-1143.

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电离辐射对Beclin1过表达和低表达MCF-7细胞模型细胞自噬和凋亡的影响及其调控机制

Effects of ionizing radiation on autophagy and apoptosis in MCF-7 cells with Beclin 1 over-and low-expressions and their regulating mechanisms

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