菲立磁标记大鼠骨髓基质细胞及自体移植后磁共振示踪的研究

Primary study on intracellular labeling of bone marrow stromal cells with Feridex and in vivo tracking after autologous transplantation with MRI

目的初步探索利用菲立磁(Feridex)和转染试剂体外磁性标记大鼠骨髓基质细胞这一方法的可行性,为将来临床上应用磁共振成像(MRI)追踪标记细胞奠定基础。方法无菌条件下行股骨取骨髓,梯度密度离心法分离获取大鼠骨髓基质细胞,使用“Feridex-多聚赖氨酸复合物(FE-PLL)”标记骨髓基质细胞,采用普鲁士蓝染色、电镜和台盼蓝排除实验等方法鉴定FE-PLL标记大鼠骨髓基质细胞的效率和细胞的活力,并对FE-PLL标记骨髓基质细胞的增殖和分化能力进行评估。将FE-PLL标记和未标记后的骨髓基质细胞分别移植入大鼠左右侧尾壳核脑内,细胞移植后1、4、7周应用MRI对脑内移植的细胞进行活体示踪,最后利用组织切片进行普鲁士蓝染色。结果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。普鲁士蓝染色显示FE-PLL标记骨髓基质细胞胞质内出现细小的蓝色铁颗粒。电镜结果显示FE-PLL标记的骨髓基质细胞胞质内含有许多包裹铁颗粒的囊泡。与正常未标记的细胞相比较,FE-PLL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像。MRI检查发现脑内移植的标记细胞在磁共振上呈明显的低信号改变,未标记细胞侧脑组织无明显的低信号改变,与组织学切片结果基本相一致。结论以上结果提示菲立磁可以用来体外标记骨髓基质细胞。

Objective To explore the feasibility of protocols using Feridex and transfection agents for in vivo magnetic labelling of bone marrow stromal cells (BMSCs) as a basis of clinical application for magnetic resonance imaging (MRI). Methods Under sterile condition, the rat BMSCs were isolated by means of density gradient centrifugation following a thighbone 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-labeled BMSCs were also investigated. Rats underwent transplantation of labeled BMSCs into the left caudate putamen and non-labeled cells were transplanted into the right part as controls.1,4 and 7 weeks after the transplantation, in vivo MRI examination was conducted on rat brains.Serial histological Prussian blue staining in sections corresponding to FE-PLL-labeled BMSCs slices seen on MRI was performed. Results BMSCs could be effectively labeled and labelling efficiency were around 99%. Prussian blue staining showed numerous blue-stained iron particles in the cytoplasm of FE-PLL-labeled BMSCs.Transmission electron microscopy of FE-PLL-labeled BMSCs revealed the presence of numerous vesicles which are filled with the electron-dense magnetic iron particles. Cell viability, proliferation and differentiation ability of labeled cells were not affected by endosomal incorporation of Feridex nanoparticles compared to the control unlabeled cells. MRI showed remarkable low signal change in the left brain transplanted with labeled BMSCs and no signal change in contra-lateral brain. MR images were correlated with histopathological Prussian staining for iron. Conclusion Feridex can be used to label BMSCs in vitro and MRI opens up the possibility of in vivo tracking of Feridex-labeled BMSCs after transplantation.