二氯化钴对腺病毒基因表达的影响  

The effect of cobalt chloride on adenovirus gene expression

作　　者：刘新建[1] 季迅达[1] 田毓华[1] 陈霞芳[1] 谢匡成[1] 吴继红[1] 张圣海[1] 徐萍[1] 李川源[2] 黄倩[1,3] 

机构地区：[1]上海交通大学附属第一人民医院中心实验室,上海200080 [2]美国科罗拉多州立大学健康科学中心放射肿瘤系,auroraco80111 [3]上海交通大学肿瘤研究所癌基因及相关基因研究国家重点实验室,上海200032

出　　处：《肿瘤》2009年第7期603-610,共8页Tumor

基　　金：国家"九七三"计划资助项目(编号:2004CB518804);国家自然科学基金资助项目(编号:30325043);国家自然科学基金资助项目(编号:30428015)

摘　　要：目的:研究二氯化钴(CoCl2)对缺氧反应元件调控E1AE1B表达的条件复制型腺病毒载体(Ad-5HRE-E1AE1B-RFP)和缺乏缺氧反应元件的复制缺陷型腺病毒载体(Ad-EGFP)在肿瘤细胞内表达和复制的影响。方法:肿瘤细胞经不同浓度的CoCl2处理后,Western印迹法检测缺氧诱导因子(hypoxia inducible factor-1α,HIF-1α)的表达;倒置荧光显微镜、FCM法及空斑形成实验等观察经CoCl2处理后,感染条件复制型腺病毒和(或)复制缺陷型腺病毒的肿瘤细胞中外源基因的表达水平和病毒复制情况;小动物成像仪观察缺氧调控的条件复制型腺病毒在肿瘤内提高复制缺陷型腺病毒表达的效率。结果:适当浓度的CoCl2(0.4和0.08μg/mL)能使胃癌细胞株(SGC7901)中HIF-1α蛋白稳定表达并积聚,可较好地模拟缺氧状态;在0.4μg/mLCoCl2作用下,Ad-5HRE-E1AE1B-RFP对肿瘤细胞的感染效率明显提高,表现为外源基因RFP阳性表达的百分率和荧光强度均明显提高,但空斑形成实验显示Ad-5HRE-E1AE1B-RFP并没有复制。0.4μg/mLCoCl2也能提高其他非缺氧调控的复制缺陷型腺病毒如Ad-EGFP、Ad-Luc的感染效率和表达水平;Ad-5HRE-E1AE1B-RFP与复制缺陷型腺病毒Ad-Luc联合注射裸鼠肿瘤能显著提高Ad-Luc的表达。结论:CoCl2能明显提高腺病毒的基因表达水平,其作用机制不仅与CoCl2诱导缺氧有关,也可能与影响基因转录有关。Objective： To investigate the effect of cobalt chloride （CoCl2） on transgene expression and viral particle titers in tumor ceils infected by conditionally replicating adenovirus expression vector with hypoxia response elements（HRE）-regulated E1AE1B expression （Ad-5HRE-E1 AE1B-RFP） and non-HRE regulated replication-deficient adenovirus expression vector （Ad-EGFP, Ad-Luc） in vitro and in vivo. Methods： Ad-5HRE-E1AEIB-RFP had five duplicated HRE and mini CMV acted as a promoter to drive E1AE1B expression and constitutive expression of RFP as reporter. The hypoxia model was optimized by exposing tumor cells to different concen- trations of COC12. The hypoxia-inducible factor 1 alpha （HIF-lct） protein expression was determined by Western blotting. Under the optimized hypoxia model, the positive expression of exogenous gene and virus replication of Ad-5HRE-E1AE1B-RFP or Ad-EGFP-infected tumor cells were examined by conversed microscopic observation, FACS analysis and plaques formation test. Furthermore, transgene expression induced by combined application of hypoxia-inducible replicative adenovirus and replication deficient adenovirus （Ad-Luc） was also evaluated by examining the lucifererse activity in xenografted tumor models in nude mice by micro PET. Results： Western blotting resuhs showed that CoCl2 at 0.4 and 0.08μg/mL could stabilize and acumulate HIF-1α protein in gastric cancer SGC7901 ceils, which could better mimic hypoxia condition. The microscopic observation and FACS analysis showed that CoCl2 at 0.4μg/mL could remarkably increase the transduction efficacy of Ad-5HRE-E1AE1B-RFP, which was verified by significant increase in the percentage of positive expression of exogenous gene RFP and fluorescence intensity. But plaques formation test showed that Ad-SHRE-E1AE1B-RFP had no replication. COCl2 0.4μg/mL augmented the tranduction efficacy and expression levels of non-HRE regulated replication deficient adenovirus Ad-EGFP and Ad-Luc. Combined intratumoral injection of Ad-SHRE-

关 键 词：胃肿瘤 腺病毒感染 基因表达调控 肿瘤 缺氧诱导因子1 二氯化钴 

分 类 号：R735.2[医药卫生—肿瘤] R73-362[医药卫生—临床医学]