大鼠骨髓源神经干细胞的Sinerem体外标记及磁共振成像研究

Sinerem labeling and MRI tracking of neural stem cells in vivo and in vitro

目的应用超小超顺磁性氧化铁(USPIO)Sinerem和转染试剂多聚赖氨酸(PLL)复合物标记大鼠骨髓源神经干细胞,初步评价磁共振成像活体示踪Sinerem标记干细胞的可行性。方法分离SD大鼠骨髓基质干细胞,体外培养诱导成骨髓源神经干细胞。将制备的Sinerem-PLL复合物以浓度Sinerem200μg/ml和干细胞共孵育培养过夜。采用普鲁士蓝染色和透射电镜确定细胞内铁的摄取、定位情况;并对细胞增殖、凋亡检测评价。体内外以SE序列T2WI与T2*WI行4.7T磁共振干细胞成像。结果该方法标记干细胞效率为95%以上,普鲁士蓝染色显示铁颗粒存在胞质中,电镜显示铁颗粒集中于内涵体和溶酶体中;该浓度时Sinerem对细胞的活性影响与未标记细胞相比差异无统计学意义(P>0.05)。标记后细胞体内外的T2WI与T2*WI信号强度明显降低。结论利用Sinerem对比剂经PLL介导标记骨髓源神经干细胞高效易行,磁共振可用于活体示踪神经干细胞。

Objective To label rat neural stem cells （NSCs） with the complex of Sinerem, the ultrasmall superparamagnetic iron oxide （USPIO）, and poly-L-lysine （PLL）, and evaluate the feasibility of tracking the labeled cells with magnetic resonance imaging （MRI） in vitro and in vivo. Methods Sinerem was incubated with PLL to obtain the complex of Sinerem-PLL. The mesenchymal stem cells （MSCs） isolated from the bone marrow of SD rats were cultured and induced to differentiate into the neural stem cells. The second-passage cells were cultured overnight with the Sinerem-PLL complex, after which Prussian blue staining and transmission electron microscopy were performed to observe the nanoparticles in the cytoplasm. Cell apoptosis assay was performed to assess the cell viability 1 day, 1 week, and 2 weeks after the labeling. Cell tracking with 4.7 MR system was carried out in vivo and in vitro using T2WI 与 T2＊WI sequences. Results The NSCs could be effectively labeled with Sinerem-PLL complex with the labeling efficiency exceeding 95%. Prussian blue staining showed numerous blue iron particles in the cytoplasm, and under transmission electron microscope, these particles accumulated in the endosomes/lysosomes, The labeling did not significantly affect the cell viability and proliferation. Remarkable low signal density changes of the labeled cells was seen on T2WI and T2＊WI in vivo and in vitro. Conclusion NSCs can be effectively labeled with Sinerem-PLL complex, and MRI can be used to track the labeled cells in vivo and in vitro.