人GDF-5重组腺病毒载体的构建及其在人间充质干细胞中的表达  

Construction of Human GDF-5 on Adenovirus Vector and Its Expression in MSCs

作　　者：程相俊[1] 刘浩[1] 薛建新[2] 张霆[3] 

机构地区：[1]四川大学华西医院骨科,成都610041 [2]四川大学华西医院肿瘤科,成都610041 [3]四川大学生物治疗国家重点实验室,成都610041

出　　处：《生物医学工程学杂志》2010年第1期120-125,共6页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(30572163)

摘　　要：本研究目的在于构建携带GDF-5基因的重组腺病毒表达载体,并用其感染人骨髓来源的间充质干细胞(MSCs)研究GDF-5的表达。从含有人GDF-5基因核心序列的质粒pCMV-SPORT6上通过PCR扩增GDF-5,将其酶切连接到穿梭质粒pAdtrack-CMV上,在BJ5183中和骨架质粒pAdeasy-1同源重组,筛选阳性克隆并酶切鉴定,线性化后磷酸钙法转染入人胚肾293细胞中包装、扩增,得到含有GDF-5基因的重组腺病毒并测定其滴度。用收集的腺病毒感染靶细胞MSCs,在基因水平检测GDF-5的表达情况。结果表明,成功构建了含有GDF-5基因的重组腺病毒载体,病毒滴度为1×109PFU/ml。重组腺病毒能有效感染MSCs并表达目的基因。通过构建该腺病毒并感染人MSCs可得到能持续一定时间表达GDF-5蛋白的MSCs,为这种转基因的MSCs进一步修复组织奠定了基础。This experimental study was aimed to construct the recombinant adenovirus vector containing human GDF-5 gene,and to use it for infecting human MSCs and detecting the expression of the gene GDF-5.The core sequence of human GDF-5 was amplified by PCR from pCMV-SPORT6,and then was cloned to pAdtrack-CMV.The linearized shuttle plasmid pAdtrack-CMV-GDF-5 was homogenously recombined with pAdeasy-1 in BJ5183.The potential clone was analyzed by restriction endonuclease digestion.The correct clone was linearized and transfected into QBI-293 cells for packing and amplifying so as to obtain adenovirus pAd-GDF-5 and identify it,while the titer was also determined by TCID50.MSCs were infected by the harvested virus,and the expression of GDF-5 was detected by RT-PCR.The recombinant adenovirus vector containing human GDF-5 gene was constructed successfully;its titer was 1×10^9 PFU/ml,and it could infect MSCs efficiently.The human MSCs infected by constructed adenovirus vector could continue expressing GDF-5 in a certain time, and the transgenic MSCs would be much potential on tissue regeneration.

关 键 词：生长分化因子5 间充质干细胞 重组腺病毒 基因治疗 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]