吉林大学学报(医学版)

• 基础研究 •    下一篇

pshuttle-Egr-1-hSmac质粒联合X线照射对乳腺癌MDA-MB-435细胞增殖的抑制作用

梁硕1,王志成1,李艳博1,2,郭彩霞2,龚守良1,林承赫3   

  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. 首都医科大学公共卫生与家庭医学学院,北京 100069;3. 吉林大学第一医院核医学科,吉林 长春  130021)
  • 收稿日期:2014-01-10 出版日期:2014-09-28 发布日期:2014-09-28
  • 通讯作者: 林承赫 E-mail:(Tel:0431-88782717,E-mail:linchh1967@163.com)
  • 作者简介:梁硕(1973-),男,吉林省长春市人,讲师,医学博士,主要从事肿瘤基因-放射治疗方面的研究。 
  • 基金资助:

    国家自然科学基金资助课题(30870747);吉林大学基本科研项目资助课题(2012)

Inhibitory effect of pshuttle-Egr-1-hSmac plasmid combined with X-ray irradiation on proliferation of breast cancer MDA-MB-435 cells

LIANG Shuo1,WANG Zhi-cheng1,LI Yan-bo1,2,GUO Cai-xia2,GONG Shou-liang1,LIN Cheng-he3   

  1. (1. Key Laboratory of Radiobiology,Ministry of Health,School of Public Health,Changchun 130021,China;2. School of Public Health and Family Medicine,Capital Medical University,Beijing 100069,China;3. Department of Nuclear Medicine,First Hospital,Jilin University,Changchun 130021,China)
  • Received:2014-01-10 Online:2014-09-28 Published:2014-09-28

摘要:

目的:构建pshuttle-Egr-1-hSmac质粒并转染人乳腺癌MDA-MB-435细胞,观察其抑制肿瘤细胞的辐射增敏作用。方法:转染pshuttle-Egr-1-hSmac质粒的MDA-MB-435细胞经过2 Gy X线照射不同时间(4、8、12、24 和48 h)和0.5 ~ 5.0 Gy X线照射后24 h收集细胞,采用RT-PCR和Western blotting法检测Smac mRNA及其蛋白表达。将细胞分为对照组、pshuttle质粒组、pshuttle-Egr-1-hSmac 质粒组、2 Gy 组、pshuttle+2.0 Gy组 和pshuttle-Egr-1-hSmac+ 2.0 Gy组,MTT法检测各组细胞增殖;克隆形成实验检测细胞存活能力;Annexin Ⅴ-FITC双染法检测细胞凋亡;PI单染法检测细胞周期。结果:对照组和pshuttle质粒组MDA-MB-435细胞中Smac mRNA无表达,而pshuttle-Egr-1-hSmac质粒组MDA-MB-435细胞Smac mRNA表达水平随时间延长逐渐升高,于24和48 h时表达水平最高;经0.5 ~ 5.0 Gy X线照射后24 h MDA-MB-435细胞Smac mRNA表达水平随照射剂量增加而逐渐增加,在2.0和5.0 Gy X线照射后Smac mRNA表达水平最高。pshuttle-Egr-1-hSmac质粒组4、8、12和24 h后Smac蛋白表达水平逐渐升高,24 h后表达水平最高。经过0、0.5、1.0、2.0和5.0 Gy X线照射后24 h Smac蛋白表达水平逐渐升高,尤其以5.0 Gy X线照射时表达水平最高。MTT法检测时程效应,2.0 Gy、pshuttle+2.0 Gy和pshuttle-Egr-1-hSmac+2.0 Gy质粒组24、48和72 h细胞A490值明显低于对照组(P<0.01);剂量效应,pshuttle-Egr-1-hSmac质粒组1.0~5.0 Gy X线照射后,MDA-MB-435细胞A490值明显低于0 Gy X线照射(P<0.05或P<0.01)。pshuttle-Egr-1-hSmac质粒组细胞存活分数明显低于对照组(P<0.01)。pshuttle-Egr-1-hSmac+2.0 Gy组细胞凋亡率明显高于2.0 Gy组(P<0.01),G0/G1期和S期细胞百分率明显低于2.0 Gy照射组(P<0.01),G2/M期细胞百分率明显高于2.0 Gy组(P<0.01)。结论:X线照射能增加pshuttle-Egr-1-hSmac质粒转染的MDA-MB-435细胞有效表达Smac mRNA及蛋白,能抑制细胞存活,且诱导G2/M期阻滞和凋亡增加;Smac基因联合放射治疗可明显增加乳腺癌细胞的放射敏感性。

关键词: Smac基因, Egr-1启动子, X射线, 基因-放射治疗, 细胞凋亡

Abstract:

Abstract:Objective
To construct the pshuttle-Egr-1-hSmac plasmid and transfect human breast cancer MDA-MB-435 cells, and to observe its radiotherapy enhancing effect on tumor cells.Methods The empty vector pshuttle and pshuttle-Egr-1-hSmac plasmid were transfected into MDA-MB-435 cells by liposomal.At different time(4,8,12,24 and 48 h) after irradiation with 2.0 Gy X-ray and 24 h after irradiation with 0.5―5.0 Gy,the total RNA and protein were collected and extracted from these cells to analyze the Smac mRNA and protein expression levels with RT-PCR and Western blotting methods.The cells were divided into control,pshuttle,pshuttle-Egr-1-hSmac,2.0 Gy irradiation group,pshuttle + 2.0 Gy irra diation and pshuttle-Egr-1-hSmac+2.0 Gy irradiation groups.MTT method was used to evalua te cell proliferation,and the cell survival ability  was measured with clone formation assay;Annexin Ⅴ/PI double staining and PI single staining were used to examine the  apoptosis and cell cycle of MDA-MB-435 cells.Results There was no Smac mRNA expression in MDA-MB-435 cells in control and pshuttle groups,but the Smac mRNA expression levels in  MDA-MB-435 cells in  pshuttle-Egr-1-hSmac plasmid group were gradually increased with the time prolongation,and reached the maximum at 24 and 48 h;the Smac mRNA expression levels in MDA-MB-435 cells  were increased gradually 24 h after irradiation of 0.5―5.0 Gy X-ray with the increasing of irradiation doses,and reached the maximum after 2.0 and 5.0 Gy irradiation.The Smac protein expr ession levels in  pshuttle-Egr-1-hSmac plasmid group were increased gradually with the time prolongation,and reached the maximum at 24 h.The Smac protein expression lervels were increased 24 h afer irradiation of 0,0.5,1.0,2.0 and 5.0 Gy X-ray,especially in 5.0 Gy group.The MTT results showed that the  A490 values  in 2.0 Gy,pshuttle+2.0 Gy and    pshuttle-Egr-1-hSmac groups 24,48,and 72 h after  irradiation were lower than th ose in control group(P<0.01);the  A490 values  of MDA-MB-435 cells in  pshuttle-Egr-1-hSmac group after 1.0-5.0 Gy X-ray irradiation were lower than those in 0 Gy group (P<0.05 or P<0.01);the survival fraction(SF) in pshuttle-Egr-1-hSmac group was lower than those in control group(P<0.01). The percentages of the cells at G0/G1 and S phase in pshuttle-Egr-1-hSmac group were lower than those in 2.0 Gy group(P<0.01),the percentage of the cells at G2/M phase was higher than that in 2.0 Gy group (P<0.01);the apoptotic rate of the cells  in pshuttle-Egr-1-hSmac group was higher than that in 2.0 Gy group (P<0.01).Conclusion X-ray irradiation can significantly increase the Smac mRNA and protein expression levels in MDA-MB-435 cells transfected with pshuttle-Egr-1-hSmac plasmid,inhibit the  cell survival rate,and  induce G2/M arrest and apoptotic increasing;Smac gene combined with radiotherapy could  significantly increase the  radiosensitivity of breast cancercells.

Key words: Smac gene, Egr-1 promoter, X-ray, gene-radiotherapy, apoptosis

中图分类号: 

  • R737.9