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

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2.0 Gy 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-E1Bp-E1B55K（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、2 Gy组、空病毒（CRAd.p）组、CRAd.p + 2 Gy组、CRAd.p-Egr1-TRAIL组和CRAd.p-Egr1-TRAIL + 2 Gy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24 h后给予2.0 Gy X射线照射，4 h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 + 2.0 Gy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；6 h后各组MDA-MB-231细胞中TRAIL蛋白表达水平开始升高，24 h后各组TRAIL蛋白表达水平达峰值，48 h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 + 2.0 Gy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2.0 Gy X射线照射可使MDA-MB-231细胞中TRAIL mRNA和蛋白表达水平升高。

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

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.