构建一种受hTERT启动子和miR-145双调控的更为安全的新型溶瘤腺病毒  

Construction of a Further Safer Conditional Replicating Adenovirus Controlled by hTERT Promoter and miR-145-regulated System

作　　者：王荣花[1] 韦芳[1] 李惠明[1] 胡冉[1] 王慧萍[1] 

机构地区：[1]上海交通大学附属第一人民医院实验中心,上海200080

出　　处：《现代生物医学进展》2014年第35期6801-6806,共6页Progress in Modern Biomedicine

基　　金：国家自然科学基金面上项目(30672440);上海市科委创新行动(13140902802)

摘　　要：目的:构建一种受人端粒酶逆转录酶(h TERT)启动子和mi R-145双调控的更加安全的条件复制型腺病毒。方法:在已构建的h TERT启动子调控早期复制必需基因E1A的条件复制型腺病毒Adzq11基础上,再将四个拷贝的mi R-145的靶序列加入到E1A的3'非翻译区,构建新型溶瘤腺病毒Adzq11-145T。应用Real-time PCR测定人非小细胞肺癌细胞A549和人胚肺成纤维细胞HLF中mi R-145的表达量;然后在这两株细胞中,分别转染Adzq11和Adzq11-145T对应的穿梭质粒及感染病毒本身,用Real-time PCR检测E1A表达量以测定调控效果;用病毒空斑单位法检测两种病毒的复制情况;用CCK8检测对A549和HLF细胞的杀伤作用。结果:构建的新型溶瘤腺病毒Adzq11-145T在HLF中复制量明显低于Adzq11,其E1A m RNA水平和杀伤效应也显著降低。而在A549中,Adzq11-145T在E1A表达、病毒复制和溶瘤能力方面较Adzq11均无显著降低。结论:成功构建出一种新的受转录和转录后水平双重调控的条件复制型腺病毒Adzq11-145T,它比仅加入h TERT启动子调控早期复制必需基因E1A的腺病毒Adzq11更加安全。Objective： To construct a safer type of conditional replicating adenovirus controlled by human telomerase reverse transcriptase（h TERT） promoter and mi R-145-regulated system. Methods： Based on oncolytic adenovirus Adzq11, whose replication was controlled by E1 A gene located downstream of tumor-specific h TERT promoter, we inserted four copies of mi R-145 complementary sequences into the 3＇-untranslated region of the E1 A gene to construct a novel oncolytic adenovirus Adzq11-145 T. The mi R-145 expression level in A549 and HLF was detected by using real-time RT-PCR. The E1 A m RNA levels post viral infected and plasmid transfected were determined by real-time RT-PCR. Adenovirus cytotoxicity assay was performed by cell counting kit（CCK8）.The viral titration was detected by Plaque Assay. Results： In normal cell HLF, the E1 A m RNA levels, the lysis activity and the replication capacity of Adq11-145 T were less significant than those of Adzq11, while didn＇t reduced in tumor cell A549. Conclusion： Compared to oncolytic adenovirus Adzq11 controlled by h TERT promoter only, the novel oncolytic adenovirus Adzq11-145 T, double controlled by h TERTpromoter and mi R-145, showed improved safety profiles.

关 键 词：人端粒酶启动子 安全性 MIR-145 溶瘤腺病毒 

分 类 号：Q78[生物学—分子生物学] R734.2[医药卫生—肿瘤]