携带TRAIL基因的条件复制型腺病毒载体的构建及其辐射诱导表达  

Construction of conditionally replicative adenovirus vector carrying TRAIL gene and its mRNA and protein expressions induced by ionizing radiation

作　　者：王宏芳[1,2] 吴嘉慧[1] 刘纯岩[3] 刘威武[1,3] 孙延红[1] 龚守良[1] 王志成[1] 刘扬[1,4] 

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]北京市疾病预防控制中心,北京100013 [3]吉林大学第二医院放射线科,吉林长春130041 [4]吉林大学公共卫生学院放射医学实验教学中心,吉林长春130021

出　　处：《吉林大学学报（医学版）》2014年第4期699-704,共6页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(30870747)

摘　　要：目的:构建携带早期生长反应基因-1(Egr-1)启动子和肿瘤坏死因子相关的凋亡诱导配体(TRAIL)基因的条件复制型腺病毒载体pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K,观察其联合2.0Gy X射线照射对人乳腺癌MDA-MB-231细胞TRAIL表达的影响。方法:以pMD18T-Egr1为模板,成功克隆Egr-1序列,将TRAIL基因置于下游,构建条件复制型腺病毒载体pShuttle-Egr1-TRAIL-hTERT-E1A-E1BpE1B55K(CRAd.pEgr1-TRAIL),病毒包装后感染细胞,给予X线照射,利用Real time PCR法检测TRAIL mRNA表达水平,ELISA法检测TRAIL蛋白表达水平。实验设对照(control)组、2Gy组、空病毒(CRAd.p)组、CRAd.p+2Gy组、CRAd.p-Egr1-TRAIL组和CRAd.p-Egr1-TRAIL+2Gy组。结果:成功构建pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K,并进行了病毒包装。以病毒滴度5感染复数(MOI)感染MDA-MB-231细胞24h后给予2.0Gy X射线照射,4h后MDA-MB-231各组细胞中TRAIL mRNA表达水平开始升高,8h后各组表达达峰值;CRAd.pEgr1-TRAIL+2.0Gy组mRNA表达水平升高最为明显,约为对照组的160倍(P<0.01),随后表达水平逐渐下降;6h后各组MDA-MB-231细胞中TRAIL蛋白表达水平开始升高,24h后各组TRAIL蛋白表达水平达峰值,48h后TRAIL蛋白表达水平下降,但仍未降至正常水平;与其他各组比较,CRAd.pEgr1-TRAIL+2.0Gy组TRAIL蛋白表达水平升高最明显(P<0.01)。结论:成功获得条件复制型腺病毒载体,联合2.0Gy X射线照射可使MDA-MB-231细胞中TRAIL mRNA和蛋白表达水平升高。Objective To construct the conditionally replicative adenovirus vector pAd-Egr1-TRAIL-hTERT-E1AE1Bp-E1B55Kcarrying early growth response gene-1（Egr1）promoter and tumor necrosis factor related apoptosis inducing ligand（TRAIL）gene,and to observe the effects of the vector combined with 2Gy irradiation on the TRAIL expression in MDA-MB-231cells.Methods Egr-1promotor sequence was cloned from pMD18T-Egr1,TRAIL was constructed the downstream of Egr1promoter,pShuttle-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K（CRAd.pEgr1-TRAIL）was constructed,after the adenovirus vector was packaged successfully,MDA-MB-231cells were infected with them and irradiated with X-rays.Real time PCR method and ELISA were used to detect the expression levels of TRAIL mRNA and protein,respectively.Six groups in the experiment were set up：control,2Gy,CRAd.p,CRAd.pEgr1-TRAIL,CRAd.p ＋ 2 Gy and CRAd.pEgr1-TRAIL ＋ 2 Gy.Results The recombinant adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K was constructed and packaged successfully.The expression level of TRAIL mRNA in MDA-MB-231cells transfected with the vector of 5MOI for24hfollowing 2.0Gy X-rays irradiation began to increase and arrived to the top 8hlater in various groups,then declined.The expression level of TRAIL protein in MDA-MB-231cells began to increase 6hafter irradiation and reached to the peak 24hlater,then declined 48hlater.There were significant differences in the expression levels of TRAIL protein between CRAd.pEgr1-TRAIL ＋ 2.0Gy and other groups at the same time point（P〈0.01）.Conclusion The recombinant adenovirus vector is obtained successfully,and the TRAIL mRNA and protein expression levels in MDA-MB-231cells can be increased significantly by the vector combined with 2.0Gy X-rays irradiation.

关 键 词：X射线 肿瘤坏死因子相关的凋亡诱导配体 条件复制型腺病毒 MDA-MB-231细胞 

分 类 号：Q782[生物学—分子生物学]