荧光素酶基因稳定表达的间充质干细胞系的构建

Establishment of luciferase-labeled mesenchymal stem cells

目的 通过表达荧光素酶的慢病毒载体感染间充质干细胞株,建立稳定表达荧光素酶的间充质干细胞系. 方法 2013年5月至2014年1月,根据gene bank查询荧光素酶基因设计引物,PCR扩增获得目的基因,体外构建质粒,构建表达荧光素酶的慢病毒载体,并感染间充质干细胞株C3H10T1/2.Puromycin浓度为2mg/ml加压筛选,鉴定.Western blotting检测荧光素酶的表达,免疫荧光观察荧光素酶在小鼠间充质细胞C3H10T1/2中的表达. 结果 Western blotting检测,结果显示此细胞表达的蛋白大小约60×10^3,表明感染病毒的C3H10T1/2细胞表达荧光素酶蛋白.免疫荧光检测,与未感染病毒的正常对照组相比,感染病毒的细胞组约100％细胞红色荧光呈阳性,表明已经构建好稳定表达荧光素酶的C3H10T1/2细胞. 结论 通过表达荧光素酶的慢病毒转染间充质干细胞,可获得荧光素酶稳定表达的间充质干细胞.转染效果确切,荧光素酶基因表达稳定.

Objective To establish luciferase-labeled mesenchymal stem cells in vitro.Methods From May,2013 to January,2014,recombinant lentiviral vectors containing luciferase gene was transfected in to mesenchymal stem cell line C3H10T1/2.Puromycin was used to filter the cells.Western-blot and immunofluorescenceimaging were used to detect the transfection efficiency.Results Western blotting analysis showed that the size of this protein in cells expressing about 60KD.Showed that infected C3H10T1/2 cells expressing luciferase protein.I mmunofluorescence,compared with the uninfected control group,virus-infected cells group of about 100% of the cells were positive for red fluorescence.Show that had built a good stable expression of luciferase C3H10T1/2 cells.Conclusion A monoclonal cell line stably and highly expressing luciferase is obtained with luciferase-labeled mesen chymal stem cells.