利用菲立磁细胞内标记恒河猴骨髓基质细胞的初步实验

Primary study on intracellular labeling of neural stem cells derived from bone marrow of rhesus monkey with Feridex

目的:探索利用菲立磁和转染试剂体外磁性标记恒河猴骨髓基质细胞的可行性,为临床上应用磁共振成像追踪标记细胞奠定基础。方法:实验于2004-04/2005-05在南方医科大学珠江医院全军神经医学研究所完成。纳入健康恒河猴3只,无菌条件下取骨髓,梯度密度离心法分离获取恒河猴骨髓基质细胞,使用“菲立磁-多聚赖氨酸复合物”标记骨髓基质细胞,采用普鲁士兰染色、电镜和锥虫蓝排除实验等方法鉴定菲立磁-多聚赖氨酸复合物标记恒河猴骨髓基质细胞的效率和细胞的活力,并对菲立磁-多聚赖氨酸复合物标记骨髓基质细胞的增殖和分化能力进行评估。结果:①菲立磁可以高效率地标记骨髓基质细胞,标记效率在99%左右。②普鲁士蓝染色显示菲立磁-多聚赖氨酸复合物标记骨髓基质细胞胞质内出现细小的蓝色铁颗粒。③电镜结果显示菲立磁-多聚赖氨酸复合物标记的骨髓基质细胞胞质内含有许多包裹铁颗粒的囊泡。④与正常未标记的细胞相比较,菲立磁-多聚赖氨酸复合物标记对骨髓基质细胞的活力、增殖和分化等能力没有明显的影响。结论:菲立磁可以用来体外标记恒河猴骨髓基质细胞。

AIM： To explore the feasibility of protocols by using Feridex and transfection agents for in vitro magnetic labeling of bone marrow stromal cells （BMSCs） as a basis of clinical application for magnetic resonance imaging （MRI）. METHODS： The experiment was carried out in the Military Institute of Neurology, Zhujiang Hospital of Southern Medical University from April 2004 to May 2005. Under the sterile condition, the bone marrow stromal cells of 3 rhesus monkeys were harvested by means of density gradient centrifugation following a bone puncture. Feridex-poly-l-lysine （FE-PLL） complexes were used to magnetically label BMSCs. The efficiency and cellular viability of FE-PLL labeled BMSCs were evaluated by Prussian blue staining, electron microscopy and trypan blue dye exclusion test. The proliferation and differentiation ability of FE-PLL labeling BMSCs were also investigated. RESULTS： ① BMSCs could be effectively labeled and the labeling efficiency were around 99%. ② Prussian blue staining showed that numerous blue stained fine particles in the cytoplasm of FE-PLL labeled BMSCs. ③ Transmission electron microscopy of FE-PLL labeled BMSCs revealed that the presence of numerous vesicles which were filled with the electron-dense magnetic iron particles. ④ Cell viability, proliferation and differentiation ability of labeled cells were not affected by endosomal incorporation of Feridex nanoparticles compared to control unlabeled cells. CONCLUSION： Feridex might be used to label BMSCs of rhesus monkey.