携带绿色荧光蛋白基因慢病毒转染的大鼠骨髓间充质干细胞的干细胞特性检测  

The stem cell characteristics of rat bone marrow mesenchymal stem cell transfected with Lentivirus-GFP

作　　者：管小俊[1] 宋琳[1] 郭雪君[1] 徐卫国[1] 

机构地区：[1]上海交通大学医学院附属新华医院呼吸内科,上海200092

出　　处：《诊断学理论与实践》2011年第6期527-530,共4页Journal of Diagnostics Concepts & Practice

基　　金：上海市科委基础研究重点项目(09JC1410700)

摘　　要：目的:研究携带绿色荧光蛋白(green fluorescent protein,GFP)基因的慢病毒转染的骨髓间充质干细胞(mesenchymal stem cell,MSC)是否仍具有干细胞的特性。方法:培养大鼠骨髓MSC,采用携带GFP基因的慢病毒转染MSC。用不同感染复数(multiplicity of infection,MOI,分别设定为5、10、25、50)转染MSC,在荧光显微镜下观察转染效果,流式细胞仪测定最佳MOI的转染效率和携带GFP的MSC(MSC-GFP)表面抗原CD表型,采用成脂肪、成骨、成软骨分化液诱导MSC-GFP分化,荧光显微镜和化学染色观察诱导分化结果。结果:MOI=25时细胞死亡少,转染效果最佳,转染效率为97%。MSC-GFP不表达CD11b/c(2%)、CD45(3%),表达CD29(100%)、CD90(99%),可向脂肪细胞、骨细胞、软骨细胞分化,符合干细胞特性。结论:携带GFP基因的慢病毒转染的骨髓MSC维持着干细胞特性。本研究结果为MSC在体内示踪研究提供理论基础。Objective To evaluate the stem cell characteristics of bone marrow mesenchymal stem cells（MSCs） transfected with lentiviral vector encoding green fluorescent protein（GFP）.Methods After isolating and culturing rat bone marrow mesenchymal stem cells,MSCs were tranfected with lentiviral vector carrying GFP（Lentivirus-GFP）at different multiplicity of infection（MOI=5,10,25,50）.Transfection efficacy was evaluated by fluorescence microscopy.Flow cytometry was used to evaluate the transfection efficiency at proper MOI and CD phenotype on the surface of MSCs carrying GFP（MSC-GFP）.Adipogenic,osteogenic,chondrogenic differentiation of MSC-GFP were detected by fluorescence microscopy and immunohistochemical stain.Results MOI = 25 was the proper dosage for MSCs transfection with fewer cell death and 97% transfection efficiency.MSC-GFP expressed positively the CD29（100%）,CD90（99%）,and negatively the CD11b / c（2%）,CD45（3%）.MSC-GFP had the potency of differentiating into fat,bone and cartilage cells.The biological characteristics of MSC-GFP did not change after transfection.Conclusions MSCs transfected with lentivirus carrying GFP do not change the stem cell characteristics,which provides a theoretical basis for the in vivo tracing study.

关 键 词：间充质干细胞 绿色荧光蛋白 慢病毒载体 机制 

分 类 号：R446.8[医药卫生—诊断学]