Egr1介导的人截短型凋亡诱导因子表达载体的构建及其在乳腺癌MCF-7细胞中的辐射诱导表达规律

J4 ›› 2012, Vol. 38 ›› Issue (3): 521-527.

Egr1介导的人截短型凋亡诱导因子表达载体的构建及其在乳腺癌MCF-7细胞中的辐射诱导表达规律

王剑锋^1,2|方芳^2,3|刘扬^2,4|吴嘉慧^2,5|龚守良²|王志成²

1. 吉林大学中日联谊医院放疗科|吉林长春 130033；2. 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；3. 吉林大学公共卫生学院营养与食品卫生学教研室,吉林长春130021；4. 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;5. 齐齐哈尔医学院公共卫生学院实验中心|黑龙江齐齐哈尔 161006

收稿日期:2011-12-27 出版日期:2012-05-28 发布日期:2012-05-28
通讯作者: 龚守良（Tel:0431-85619443，E-mail:gongsl@163.com）;王志成（Tel:0431-85619443，E-mail:zhicheng@jlu.edu.cn） E-mail:gongsl@163.com;zhicheng@jlu.edu.cn
作者简介:王剑锋（1978-）|男|吉林省永吉县人|主治医师|医学博士|主要从事电离辐射生物效应及肿瘤放射治疗的研究。
基金资助:
国家自然科学基金资助课题（30970681）；吉林大学科学前沿与交叉学科创新项目资助课题（201103258）

Construction of Egr1-mediated human truncated apoptosis inducing factor expression vector and its expression regularity  induced by radiation in breast cancer MCF-7 cells

WANG Jian-feng^1,2,FANG Fang^2,3,LIU Yang^2,4,WU Jia-hui^2,5,GONG Shou-liang²,WANG Zhi-cheng²

1. Department of Radiotherapy,China-Japan Union Hospital,Jilin University,Changchun 130033,China; 2. Key Laboratory of Radiobiology,Ministry of Health,School of Public Health,Jilin University,Changchun 130021,China;3. Department of Nutrition and Food Hygiene,School of Public Health,Jilin University,Changchun 130021,China;4. Radiation Medicine Teaching Experiment Center,School of PublicHealth,Jilin University,Changchun 130021;5. Experimental Center,School |of Public Health,Qiqihar Medical University,Qiqihar 161006,China

Received:2011-12-27 Online:2012-05-28 Published:2012-05-28

摘要/Abstract

摘要：

目的: 克隆人截短型凋亡诱导因子（AIF）cDNA序列，并构建早期生长反应因子1（Egr1）介导的重组表达载体pcDNA3.1-Egr1-AIFΔ1-480（pEgr1-AIFΔ1-480），观察其在人乳腺癌MCF-7细胞中的辐射诱导表达规律。方法: 以人白血病Jurkat细胞mRNA为模板，RT-PCR法扩增获得人AIFΔ1-480，与pMD18T载体连接后行全自动测序，限制性内切酶切取pMD19T-Egr1中Egr1片段，并利用基因重组技术构建Egr1启动子介导的人AIFΔ1-480表达质粒pEgr1-AIFΔ1-480。实验分为对照组、 pcDNA3.1组、pAIFΔ1-480组和 pEgr1-AIFΔ1-480组。各组质粒分别转染MCF-7细胞，Western blotting法检测AIF及AIFΔ1-480蛋白的辐射诱导表达时程-效应（2.0 Gy照射后0、2、4、12、24和48 h）和剂量-效应（0、0.2、0.5、1.0、2.0、5.0 Gy照射后24 h）规律。结果: 经测序证实，RT-PCR获得的人AIFΔ1-480基因与预期一致，pEgr1-AIFΔ1-480经PCR和酶切鉴定完全正确；各组MCF-7细胞经2.0 Gy照射后0～48 h，AIF蛋白在各组中均有表达，从4 h开始显著增加，4、12、24和48 h各组AIF表达与0 h组比较差异有统计学意义（P<0.05），48 h达到最大；而在pAIFΔ1-480和pEgr1-AIFΔ1-480组，AIFΔ1-480从2 h开始表达，4、12、28和48 h各组AIFΔ1-480表达与2 h比较差异有统计学意义（P<0.05），24 h达到峰值；而且24和48 h时pEgr1-AIFΔ1-480组AIFΔ1-480表达较pAIFΔ1-480组显著增加（P<0.05）。各组MCF-7细胞经0～5.0 Gy照射后24 h，各组均有AIF蛋白表达，而AIFΔ1-480只在pAIFΔ1-480和pEgr1-AIFΔ1-480组表达，二者均随着剂量增加而增加，0.2、0.5、1.0、2.0和5.0 Gy照射后各组AIF表达与0 Gy比较差异有统计学意义（P<0.05），在5.0 Gy照射时达到最大，相同照射剂量pEgr1-AIFΔ1-480组AIFΔ1-480表达高于pAIFΔ1-480组。结论: 成功构建Egr1介导的人截短型AIF重组表达载体pEgr1-AIFΔ1-480， AIF和AIFΔ1-480蛋白在MCF-7细胞中的表达随照射时间延长和照射剂量增加而增加。

关键词: Egr1；截短型凋亡诱导因子；辐射；乳腺肿瘤；基因重组

Abstract:

Abstract:Objective To clone human truncated apoptosis inducing factor (AIF) cDNA sequence,and to construct early growth response 1 (Egr1)-mediated recombinant expression vector pcDNA3.1-Egr1-AIFΔ1-480 (pEgr1-AIFΔ1-480),and to observe its regularity induced by radiation in human breast cancer MCF-7 cells.Methods The total mRNA extracted from human leukemia jurkat cells used as template,and the human AIFΔ1-480 was acquired by RT-PCR,and it was linked to pMD18T vector for sequencing.Egr1 fragment was digested from pMD19T-Egr1 by restrictive enzyme,and the Egr1-mediated expression plasmid pEgr1-AIFΔ1-480 was constructed by gene recombination.There were control group,pcDNA3.1 group,pAIFΔ1-480 group and pEgr1-AIFΔ1-480 group in the experiment. After the plasmids in various groups were transfected into human breast cancer MCF-7 cells,the AIF and AIFΔ1-480 protein expression time-effect (0,2,4,12,24 and 48 h after 2.0 Gy irradiation) and dose-effect (24 h after 0,0.2,0.5,1.0,2.0 and 5.0 Gy irradiation) regularity were measured by Western blotting method.Results The sequencing results showed that the AIFΔ1-480 acquired by RT-PCR was consistent with the sequence expected，the pEgr-AIFΔ1-480 was confirmed by PCR and restrictive enzyme digestion.After 0-48 h the MCF-7 cells were irradiated by 2.0 Gy,and the AIF protein expressed in the cells in each group,and it increased significantly from 4 h and the AIF expressions in 4,12,24 and 48 h groups were higher than that in 0 h group(P<0.05),and it reached to maximum value at 48 h.But the AIFΔ1-480 protein expressed in the cells in pAIFΔ1-480 and pEgr1-AIFΔ1-480 groups from 2 h (P<0.05),and it reached to peak value at 24 h.The AIFΔ1-480 expressions in pEgr1-AIFΔ1-480 group were higher than those in pAIFΔ1-480 group at 24 and 48 h (P<0.05).After the MCF-7 cells were irradiated by 0-5 Gy for 24 h,the AIF protein expressed in the cells in each group,but the AIFΔ1-480 protein expressed merely in the cells in pAIFΔ1-480 and pEgr1-AIFΔ1-480 groups.The AIF and AIFΔ1-480 expressions were increased with the dose increasing,the AIF expressions irradiated with 0.2，0.5，1.0 and 5.0 Gy were higher than that with 0 Gy irradiation.It reached to maximum value by 5.0 Gy irradiation and the AIFΔ1-480 expression in pEgr1-AIFΔ1-480 group was higher than that in pAIFΔ1-480 group.Conclusion The human truncated AIF expression recombinant vetor pEgr1-AIFΔ1-480 is successfully constructed,and the AIF and AIFΔ1-480 protein expressions increased with time prolongation and dose increasing after irradiation in MCF-7 cells.

Key words: early growth response gene 1;truncated AIF;radiation;breast neoplasms;gene recombination

中图分类号:

R737.9

王剑锋|方芳|刘扬|吴嘉慧|龚守良|王志成. Egr1介导的人截短型凋亡诱导因子表达载体的构建及其在乳腺癌MCF-7细胞中的辐射诱导表达规律[J]. J4, 2012, 38(3): 521-527.

WANG Jian-feng,FANG Fang,LIU Yang,WU Jia-hui,GONG Shou-liang,WANG Zhi-cheng. Construction of Egr1-mediated human truncated apoptosis inducing factor expression vector and its expression regularity  induced by radiation in breast cancer MCF-7 cells[J]. J4, 2012, 38(3): 521-527.

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