Sinerem标记大鼠骨髓基质细胞及移植后活体示踪研究

Primary study on labeling of bone marrow stromal cells with Sinerem and in vivo tracking

目的初步探索利用欣诺(Sinerem)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Sinerem-多聚赖氨酸复合物(SE-PLL)标记骨髓基质细胞,采用普鲁士兰染色、电镜和台盼蓝排除实验等方法鉴定SE-PLL标记大鼠骨髓基质细胞的效率和细胞的活力,并对SE-PLL标记骨髓基质细胞的增殖和分化能力进行评估。将SE-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、7w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兰染色。结果Sinerem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SE-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SE-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,SE-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响。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一致。结论以上结果提示Sinerem可以用来体外标记骨髓基质细胞,利用MRI技术可以对脑内移植后的标记细胞进行初步的活体追踪。

Objective To explore the feasibility of protocols using Sinerem and transfection agents for in vivo magnetic labeling of bone marrow stmmal cells （BMSCs） as a basis of the clinical application of MRI. Methods Under the sterile condition, the rat bone marrow stromal cells were harvested by means of density gradient centrifugation following a thighbone puncture. Sinerem-poly-L-lysine （SE-PLL） complexes were used to label BMSCs magnetically. The efficiency and cellular viabihty of SE-PLL labeled BMSCs were evaluated by Prussian blue staining, electron microscopy, and trypan blue dye exclusion test. The proliferation and differentiation ability of SE- PLL labeling 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. In vivo MRI examination was conducted on rat brains 1,4, and 7 w after the transplantation. Serial histological Prussian blue staining in sections corresponding to SE-PLL labeled BMSCs slices showed on MRI was performed. Results BMSCs could be effectively labeled and labeling efficiency was about 99%. Prussian blue staining showed numerous blue stained fine particles in the cytoplasm of SE-PLL labeled BMSCs. Transmission electron microscopy of SE-PLL labeled BMSCs revealed the presence of numerous vesicles which were filled with the electron-dense magnetic iron particles. Cell viability, proliferation and differentiation ability of labeled cell were not affected by endosomal incorporation of Sinerem nanoparticles compared to that of unlabeled cells in control. MRI showed remarkable low signal in the left brain transplanted with labeled BMSCs and no signal change in contralateral brain. MRI was correlated with histopathological Prussian staining for iron. Conclusion The results suggest that Sinerem might be used to label BMSCs in vitro which makes it a possibility of in vivo tracking of Sinerem labeled BMSCs after transplantation.