携人胰岛素基因的“可控”型腺病毒载体的构建与初步验证  

Construction and preliminary validation of a new "Controllable" recombinant adenovirus vector carrying human insulin gene

作　　者：杨丽斌 斯朗扎西[2] 李元朝[3] 何威[3] 王儒鹏[3] 周春丽[3] 张斌[3] 

机构地区：[1]乌鲁木齐市友谊医院内分泌科 [2]中国人民解放军77563部队61分队 [3]中国人民解放军第三军医大学附属新桥医院皮肤风湿免疫科

出　　处：《实用医学杂志》2015年第11期1744-1747,共4页The Journal of Practical Medicine

基　　金：国家自然科学基金资助项目(编号:31000455)

摘　　要：目的:设计并构建一种"可控"型腺病毒载体,该腺病毒载体在加入诱导剂后能自行消亡,从而解决其激发宿主免疫及产生复制型腺病毒等问题,为临床提供一种更安全的外源基因载体。方法:以传统Ad EasyTM腺病毒载体系统为基础,将Cre-Lox P定点敲除系统的Cre酶基因安置在Tet-On诱导表达系统的下游,而在穿梭质粒右臂基因组上下游分别插入一个Lox P位点,构建成"可控"型腺病毒穿梭质粒,并插入全长人胰岛素基因,感染大鼠骨髓间充质干细胞,观察诱导剂加入前后细胞荧光蛋白表达及人胰岛素分泌情况。结果:成功构建携全长人胰岛素基因的"可控"型重组腺病毒Ad Easy N/INS,转染QBI-293A细胞和大鼠骨髓间充质干细胞后,均可观察到绿色荧光的表达,在加入诱导剂Dox后,荧光细胞数/总细胞数的比值及胰岛素水平均显著降低。结论:成功构建了携带人胰岛素全长基因的新型"可控"型腺病毒载体,该新型重组腺病毒可像传统载体一样感染BMCs,且能加入诱导剂后迅速降解,实现了外源基因载体的可控性。Objective To design and construct a ＂controlled＂ adenovirus vector in degradating by itself after induction for solving the problem of stimulating host immune and producing replication adenovirus and providing a secure exogenous gene vectors for clinical practice. Methods Based on the traditional adenovirus vector AdEasy^TM system, we inserted the Cre gene which belongs to Cre-LoxP system into the downstream of Tet-On inducible expression system. Two LoxP sites were inserted into two sides of the shuttle plasmid＇s right arm genome. Then, the full-length human insulin gene was inserted HindIII enzyme site. After the recombinant adenovirus infected the rat bone marrow-derived mesenchymal stem cells, fluorescent protein expression and insulin secretion were detected before or after induction by Dox. Results A new ＂controlled＂ recombinant adenovirus vector carrying human insulin gene was constructed successfully, and was named AdEasyN/INS. After the transfection of this new vector into QBI-293A cells and rat bone marrow mesenchymal stem cells, green fluorescent protein could be observed. After induction by Dox, both of the ratio of fluorescent cells/total cell and the levels of insulin significantly decreased. Conclusion Construction and preliminary validation of a ＂controlled＂ recombinant adenovirus vector carrying human insulin gene is constructed successfully, it could infect rat bone marrow mesenchymal stem cells, and degradate by itself after Dox induction, realize the controllability of exogenous gene carrier.

关 键 词：“可控”型腺病毒载体 人胰岛素 降解 

分 类 号：R450[医药卫生—治疗学]