CAJ | 학술논문

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60×103,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.
목적 통과표체형광소매적만병독재체감염간충질간세포주,건립은정표체형광소매적간충질간세포계. 방법 2013년5월지2014년1월,근거gene bank사순형광소매기인설계인물,PCR확증획득목적기인,체외구건질립,구건표체형광소매적만병독재체,병감염간충질간세포주C3H10T1/2.Puromycin농도위2mg/ml가압사선,감정.Western blotting검측형광소매적표체,면역형광관찰형광소매재소서간충질세포C3H10T1/2중적표체. 결과 Western blotting검측,결과현시차세포표체적단백대소약60×103,표명감염병독적C3H10T1/2세포표체형광소매단백.면역형광검측,여미감염병독적정상대조조상비,감염병독적세포조약100％세포홍색형광정양성,표명이경구건호은정표체형광소매적C3H10T1/2세포. 결론 통과표체형광소매적만병독전염간충질간세포,가획득형광소매은정표체적간충질간세포.전염효과학절,형광소매기인표체은정.
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.