超顺磁性氧化铁对大鼠神经干细胞生物学活性的影响

Effect of superparamagnetic iron oxide labeling on neural stem cell survival and proliferation

目的初步研究利用超顺磁性氧化铁标记大鼠骨髓基质细胞来源的神经干细胞及其对细胞活力、增殖等生物学活性的影响,并确定最佳标记浓度。方法无菌条件下取大鼠股骨骨髓,梯度密度离心法分离得到骨髓基质细胞。体外培养、诱导成神经干细胞,使用不同终浓度的(6.25、12.5、25、37.5、50μg/ml)Ferumoxides(超顺磁性氧化铁)标记神经干细胞,采用普鲁士蓝染色、MTT法、流式细胞仪、透射电镜等方法鉴定Ferumoxides标记神经干细胞的效率及对细胞活力、增殖等生物学特性的影响。结果Ferumoxides可以高效率标记神经干细胞,标记效率在90%以上。普鲁士蓝染色显示标记的神经干细胞胞质内存在细小蓝色铁颗粒,细胞呈淡蓝至深蓝色,颜色的深浅与所加Ferumoxides的剂量成正相关。电镜结果显示标记的神经干细胞胞质内含有许多包裹铁颗粒的囊泡,主要集中在胞体上。当Ferumoxides终浓度高于25μg/ml时,Ferumoxides颗粒在细胞内聚集成团块状,量较多,在一定程度上影响了细胞超微结构的观察;低于25μg/ml时,Ferumoxides颗粒散布于细胞胞质内,并随着浓度的降低,Ferumoxides颗粒在胞质内分布越分散,量越少。MTT及流式细胞仪结果显示Ferumoxides终浓度大于25μg/ml时则对细胞活力、增殖、凋亡有一定影响,小于等于25μg/ml时对细胞活力、增殖无明显影响。结论利用Ferumoxides在体外标记神经干细胞是一种可行的实验方案,其最佳终浓度为25μg/ml。

Objective To study the effect of superparamgnetic iron oxides （ferumoxides） on the survivaland proliferation of neural stem cells （NSCs） and determine the optimal ferumoxides concentration for labeling. Methods Bone marrow stromal cells （BMSCs） were obtained from rat femoral marrow and cultured in vitro to induce their differentiation into NSCs. Ferumoxides labeling of the NSCs was performed with different final concentrations of ferumoxides, and the labeling efficiency and viability of the labeled NSCs were evaluated by Prussian blue staining, MTT assay, flow cytometry and transmission electron microscope. Results The NSCs could be effectively labeled with ferumoxides with a labeling efficiency of around 90%. Prussian blue staining showed numerous fine granules with blue staining in the cytoplasm of the labeled NSCs, and the intensity of the blue staining was in positive correlation with the ferumoxide concentration for labeling. Transmission electron microscopy of the labeled NSCs revealed the presence of numerous vesicles spreading in the cytoplasm and filled with electron-dense magnetic iron particles. The ferumoxides vesicles increased with the labeling concentration of ferumoxides, and at the final concentration exceeding 25 μg/ml, ferumoxides vesicles in the NSCs gave rise to conglomeration which hampered observation of the cellular ultrastructure by transmission electron microscope. The results of flow cytometry and MTT assay demonstrated that the cell viability, proliferation, differentiation and apoptosis of the labeled cells were affected by ferumoxides at the concentration above 25 μg/ml, but such effects could be minimal at lower concentrations. Conclusion Ferumoxides might be feasible for in vitro labeling of the NSCs with the optimal concentration of 25 μg/ml.