【摘要】 背景:超顺磁性氧化铁标记可以活体示踪干细胞在动物体内迁徙情况,荧光活性染料DiI对细胞活力和增殖分化影响较小,适合标记和示踪细胞。目的:观察超顺磁性氧化铁及DiI双标骨髓间充质干细胞的效果和安全性。方法:无菌条件下分离大鼠双下肢,低糖DMEM培养液冲出骨髓,运用全骨髓贴壁法体外分离骨髓间充质干细胞,利用差速贴壁原理对细胞进行纯化扩增。对分离纯化并传代培养的大鼠骨髓间充质干细胞标记超顺磁性氧化铁,细胞移植前进行DiI标记。结果与结论:原代培养8-10 d后可分离得到骨髓间质干细胞,传代周期为三四天,超顺磁性氧化铁-DiI标记率近100%,普鲁士蓝染色显示蓝色铁颗粒位于骨髓间质干细胞胞质内,荧光显微镜下胞浆内示红色荧光,锥虫蓝拒染法检测标记和未标记活细胞数目均在95%左右,说明在含超顺磁性氧化铁-DiI的培养基中骨髓间充质干细胞可继续增殖,细胞形态无明显变化。以上结果显示超顺磁性氧化铁-DiI可安全有效地标记骨髓间质干细胞。更多还原

【Abstract】 BACKGROUND: Superparamagnetic iron oxide(SPIO) labeling can trace the migration of stem cells in vivo, and the fluorescent DiI dye is suitable for marking and tracing cells because of its less influence on cell viability, proliferation and differentiation. OBJECTIVE: To observe the effect and safety of SPIO and fluorescent DiI dye to double label bone marrow mesenchymal stem cells. METHODS: The bilateral lower limbs of rats were isolated sterilely. Bone marrow was obtained by rinsing using low-glucose DMEM. Bone marrow mesenchymal stem cells were isolated by the whole bone marrow adherence method and purified by differential attachment method. Purified cells were dual-labeled with SPIO particle and fluorescent DiI dye. RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells could be separated at 8-10 days after primary culture and the subculturing cycle was 3-4 days. Rat bone marrow mesenchymal stem cells could be effectively labeled with SPIO-DiI and the labeling efficiency was almost 100%. Blue irons contained in intracytoplasmatic vesicles could be observed clearly with Prussian blue staining, and the fluorescence microscopy showed red fluorescence at cytoplasm. Survival and apoptosis percentages obtained by MTT analysis were similar among labeled and unlabeled bone marrow mesenchymal stem cells that were both about95%.These findings indicate that the rat bone marrow mesenchymal stem cells could be efficiently labeled with SPIO-DiI to construct a nano-bioprobe, without significant changes in morphology, viability and proliferation.更多还原