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  

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

作　　者：梁硕[1] 王志成[1] 李艳博[1,2] 郭彩霞[2] 龚守良[1] 林承赫[3] 

机构地区：[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

出　　处：《吉林大学学报（医学版）》2014年第5期913-919,共7页Journal of Jilin University：Medicine Edition

基　　金：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)

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 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 irradiation and pshuttle-Egr-1-hSmac＋2.0 Gy irradiation groups.MTT method was used to evaluate 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 expression 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 those in control group（P〈0.01）;the A490 values of MDA-MB-435 cells in pshuttle-Egr-1-hSmac

关 键 词：SMAC基因 EGR-1启动子 X射线 基因-放射治疗 细胞凋亡 

分 类 号：R737.9[医药卫生—肿瘤]