绿色荧光蛋白转基因大鼠骨髓间充质干细胞的分离鉴定

Isolation and identification of bone marrow mesenchymal stem cells from transgenic rats with green fluorescent protein gene

背景:转基因动物提取的间充质干细胞自身携带绿色荧光蛋白,与传统病毒、质粒转染相比,能在活细胞中稳定地表达,可较快筛选其修饰的细胞。目的:观察绿色荧光蛋白转基因大鼠骨髓间充质干细胞的生物学特性。方法:取2周龄的绿色荧光蛋白转基因大鼠双侧长骨骨髓,采用全骨髓贴壁法分离培养骨髓间充质干细胞。流式细胞术分析第5代绿色荧光蛋白阳性骨髓间充质干细胞的细胞表型,并分别加入成骨及成脂条件培养液进行体外多向诱导分化,采用茜素红钙盐染色和油红O染色进行鉴定。结果与结论:成功获得了稳定表达绿色荧光蛋白的骨髓间充质干细胞,流式细胞仪检测细胞表达CD90和CD105,不表达或弱表达CD14和CD45。经成骨诱导3周后茜素红染色可见有橘红色钙盐沉积,经成脂诱导3周后油红O染色见红色的脂滴。结果表明稳定表达的绿色荧光蛋白未影响到骨髓间充质干细胞的多向分化潜能,可作为良好的示踪因子。

BACKGROUND：Mesenchymal stem cells from transgenic animals carry green fluorescent protein that can be stably expressed in viable cells and can be used to rapidly screen modified cells in comparison with traditional virus and plasmids. OBJECTIVE：To study the biological features of bone marrow derived stromal cells from transgenic rats with green fluorescent protein gene. METHODS：Bone marrows were isolated from the bilateral long bones of 2-week-old transgenic rats with green fluorescent protein gene, and cultivated in conditioned culture medium to obtain bone marrow mesenchymal stem cells with green fluorescent protein gene. The passage 5 bone marrow mesenchymal stem cells with green fluorescent protein gene were analyzed by flow cytometry to detect cellsurface molecules, and were induced in calcium or adipogenic induction medium. After calcium induction, the alizarin red staining was performed to test the formation of calcium concentration. After adipogenic induction, bone marrow mesenchymal stem cells were detected with oil red O staining. RESULTS AND CONCLUSION：Bone marrow mesenchymal stem cells, stably expressing green fluorescent protein gene, were obtained successful y from the transgenic rat with green fluorescent protein gene. The cells expressed CD90, CD105 strongly, but did not express or weakly expressed CD14 andCD45. After 3 weeks of calcium induction, jacinth calcium salts could be detected in the cells by alizarin red staining. At 3 weeks of adipogenic induction, the cells showed positive oil red O staining. The green fluorescent protein gene, as report＆amp;nbsp;gene, exerts no effect on the cellsurface molecules and multilineage potential of bone marrow mesenchymal stem cells, which can be used as an efficient tracer.