脐血间充质干细胞磁探针标记和MR成像研究

Magnetic particle labeling and in vitro MR imaging of mesenchymal stem cells from human umbilical cord blood

目的 应用磁性氧化铁纳米粒子和多聚赖氨酸 (Poly L Lysine ,PLL)的偶联物Fe2 O3 PLL ,体外标记人脐血间充质干细胞 (mesenchymalstemcells,MSCS) ,磁共振进行标记细胞成像。方法 制备Fe2 O3 PLL ,分离人脐血MSCS 进行纯化并传代培养 ,标记Fe2 O3 PLL ,普鲁士蓝染色显示细胞内铁 ,四氮噻唑蓝 (MTT)比色试验评价不同浓度Fe2 O3 PLL标记MSCS 后对细胞生长状况的影响 ,Annexin/碘化吡啶 (PI)双染色法检测细胞凋亡 ,应用MR的T1WI、T2 WI、T2 WI3个序列进行细胞群成像。结果 普鲁士蓝染色清晰显示蓝色铁颗粒位于MSCS 胞质内 ,MTT比色试验示 5～ 2 0 0 μg/ml等7个铁浓度组 ,Fe2 O3 PLL标记后细胞的光吸收值与未标记MSCS 者比较 ,各组间总体比较差异无统计学意义(Kruskal Wallis秩和检验 ,χ2 =10 35 ,P =0 17)。标记MSCS 时 ,铁浓度采用 2 0 μg/ml较合适。标记MSCS、未标记MSCS 者晚期凋亡及坏死细胞分别为 5 4 3%、2 95 % ,早期凋亡细胞分别为 9 93%、10 14 %。在MR的T1WI、T2 WI、T2 WI中 ,标记 1× 10 6个MSCS 者、标记 5× 10 5个MSCS 者较未标记MSCS 者均有信号降低改变 ,且前者的降低率大于后者 ,3个序列中以T2 WI的信号强度变化率最大 ;标记 1× 10 6个MSCS 者 ,标记后培养

Objective To label human umbilical cord blood mesenchymal stem cells (MSC S) by combining Poly-L-Lysine (PLL) with magnetic iron oxide, and to obtain 1.5 T MR images. Methods PLL was combined with magnetic iron oxide into Fe 2O 3-PLL.Human umbilical cord blood MSC S were isolated, purified, expanded, and then incubated with Fe 2O 3-PLL.Prussian blue stain was performed for showing intracellular iron.For evaluating the toxicity and proliferation of various concentrations of Fe 2O 3-PLL labeled MSC S, MTT assay was assessed.The cells apoptosis rate was determined by AnnexinV / PI double staining method.The cells underwent MR imaging with T 1WI, T 2WI, and T 2*WI. Results Iron-containing intracytoplasmic vesicles could be observed clearly with Prussian blue staining.Among various concentrations of Fe 2O 3-PLL labeled MSC S and unlabeled cells, MTT value of light absorption had no statistical significant difference (Kruskal-Wallis test, χ2=10.35, P=0.17).20 μg/ml of iron was the suitable concentration for incubating cells.The early apoptotic cells (AnnexinV-FITC positive / PI negative) of labeled and unlabeled MSC S were 9.93%, 10.14%, respectively, and late apoptotic cells (AnnexinV-FITC positive / PI positive) were 5.43%, 2.95%, respectively.The signal intensity decrease for 1×106 and 5×105 labeled cells compared with that for unlabeled cells was demonstrated on T 1WI, T 2WI, and T 2*WI.The percentage change in signal intensity (ΔSI) was larger for 106 labeled cells than that for 5×105 labeled cells especially on T 2*WI.After 8 days’ culture, ΔSI for 106 labeled MSC S decreased compared with that for the same cells 7 days ago.Conclusion The human umbilical cord blood MSC S can be labeled with Fe 2O 3-PLL without significant change in viability and apoptosis.The labeled MSC S can be imaged with standard 1.5 T MR equipment.