逆转录病毒介导的靶向人Slingshot-1L基因稳定转染人骨髓间充质干细胞系的建立  

Establishment of stable transfected human marrow mesenchymal stem cell line Slingshot-1L mediated by retroviral

作　　者：张慕蕊[1] 王岩[2] 马英智[1] 赵东旭[1] 杨旭芳[1] 李玉林[1] 

机构地区：[1]吉林大学白求恩医学院病理生物学教育部重点实验室,吉林长春130021 [2]吉林大学中日联谊医院临床基因诊疗中心

出　　处：《中国老年学杂志》2010年第3期330-332,共3页Chinese Journal of Gerontology

基　　金：国家863重大专项(2004AA205020);国家自然科学基金资助项目;吉林省科技厅重大项目(20076023)

摘　　要：目的建立过表达该基因的人骨髓间充质干细胞(hMSCs)稳定转染细胞系。方法采用密度梯度离心法联合贴壁培养法分离、纯化hMSCs。应用脂质体法将Slingshot-1L重组逆转录病毒载体pLNCX-Slingshot-1L转染PA317包装细胞,G418筛选,滴度测定。收集病毒上清感染第2代hMSCs,G418筛选,挑选阳性克隆,扩增培养。结果采用密度梯度离心法联合贴壁培养法成功分离、纯化了hMSCs,应用pLNCX-Slingshot-1L重组逆转录病毒载体转染PA317包装细胞,获得滴度为5×108CFU/L的病毒上清。收集病毒上清感染hMSCs,获得了过表达Slingshot-1L基因的hMSCs。结论应用Slingshot-1L重组逆转录病毒载体,成功建立了过表达该基因的hMSCs稳定转染细胞系。Objective To establish stable transfected human marrow mesenchymal stem cells （hMSCs） line by retroviral vector encoding Slingshot-1Lto abtain viral supernatant with high viral titers. Methods hMSCs were isolated and purified from the bone marrow of human by density gradient centrifugation and adhering to the culture dishes. The plasmid pLNCX-Slingshot-1L was transfected into packaging cell PA317 by lipofectamine. The transfectants were selected by G418 and viral titers were analyzed. Virals supernatant were collected to infect hMSCs in the second passage,selected by G418. Results Virals supernatant with high viral titers were obtained by retroviral vector encoding Slingshot-1L,virals supernatant were collected to infect hMSCs,and stable transfected hMSCs line was established. Conclusions The stable transfected hMSCs line over express Slingshot-1L mediated by retroviral was established successfully.

关 键 词：逆转录病毒 稳定转染 干细胞 

分 类 号：Q782[生物学—分子生物学]