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目的:为了追踪评价干细胞修复组织缺损时干细胞龛的分子作用机制,培养绿色荧光蛋白转基因小鼠的骨髓间充质干细胞,并对其进行鉴定和诱导分化。方法:取3-4周龄gfp小鼠股骨全骨髓,ACK裂解红细胞后采用贴壁法进行体外培养和传代,荧光显微镜下观察贴壁细胞荧光状态。运用流式细胞术检测干细胞标记物,通过体外诱导观察其成骨成脂能力。结果:原代培养的Gfp骨髓间充质干细胞,能多次传代,细胞的荧光仍保持原有状态,未发生淬灭。流式细胞术鉴定CD29、CD90表达阳性,CD45表达阴性。成骨诱导3周后骨髓间充质干细胞分化为成骨细胞,茜素红S染色有橘红色磷酸盐胞外基质沉积并保持原有的荧光强度;成脂诱导2周后,细胞形态增大,油红O染色可见胞质内大量橙红色脂肪空泡,分化的细胞仍保持原有的荧光强度。结论:贴壁培养的Gfp骨髓间充质干细胞不仅荧光表达稳定,而且具有干细胞自我更新增殖和多向分化的干细胞特性,可以做为研究干细胞龛的优质示踪干细胞源。
OBJECTIVE: To track the molecular mechanism of stem cell niche in the repair of tissue defect by stem cells, bone marrow mesenchymal stem cells of green fluorescent protein transgenic mice were cultured and identified and induced to differentiate. METHODS: Whole bone marrow of femurs of 3-4 week-old gfp mice was stained with ACK. The erythrocytes were lysed by ACK and then cultured and passaged by adherent method. Fluorescence microscopy was used to observe the fluorescence of adherent cells. Stem cell markers were detected by flow cytometry and their osteogenic and adipogenic capacity was observed by in vitro induction. Results: Primary cultured Gfp bone marrow mesenchymal stem cells can pass through many times, and the fluorescence of the cells remained the original state without quenching. Flow cytometry identified CD29, CD90 expression was positive, CD45 expression was negative. Bone marrow-derived mesenchymal stem cells differentiated into osteoblasts after osteogenic induction for 3 weeks. Alizarin red S staining deposited the orange-red phosphate extracellular matrix and maintained the original fluorescence intensity. After 2 weeks of adipogenic induction, the cell morphology was increased , Oil red O staining shows a large number of cytoplasm orange red fat vacuoles, differentiated cells still maintain the original fluorescence intensity. Conclusion: Adherent Gfp bone marrow mesenchymal stem cells not only have stable fluorescence, but also have stem cell self-renewal proliferation and multi-directional differentiation of stem cell characteristics, which can be used as a good tracer stem cell niche source.