成人外周血源性血管内皮祖细胞的磁化标记及体外MRI

Magnetic labeling human peripheral blood endothelial progenitor cells and MRI in vitro

目的 探讨磁性纳米材料低分子量聚乙烯亚胺修饰的超顺磁性氧化铁（Alkyl-PEI2k/SPIO）标记成人外周血源性血管内皮祖细胞（EPCs）的标记效率及7.0 T MR体外成像效果.方法 密度梯度离心贴壁培养EPCs.Alkyl-PEI2k/SPIO（Fe终质量浓度7μg/ml）标记EPCs,普鲁士蓝染色检测标记率;以CCK-8实验、Transwell迁移实验及Matrigel成管实验分别检测标记后细胞的生长、增殖活性,迁移能力及体外成管能力;将Alkyl-PEI2k/SPIO标记的EPCs（0、5×10^3、1×10^4、5× 10^4、1×10^5、2.5×10^5、5×10^5、1× 10^6和1.5×10^6个）制成琼脂糖凝胶细胞模型进行7.0 T MR成像.对数据行两样本t检验和配对t检验.结果 EPCs标记率达100％,标记后EPCs的含铁量为（6.062±0.050） pg/细胞;各时间点标记和未标记细胞的吸光度间差异无统计学意义（均P＞0.05）;SPIO标记组[（80.6±8.0）个]和未标记组EPCs[（77.6±4.6）个]迁移细胞数差异无统计学意义（P＞0.05）;SPIO标记组成管数量[（9.0±1.0）个]和未标记组EPCs成管数量[（9.4±1.5）个]相当（P＞0.05）.EPCs琼脂糖凝胶模型MRI结果显示,T2、T2＊图像信号强度和弛豫时间随细胞数目的增加而减低,不同细胞数凝胶模型T2和T2＊间的信号强度、弛豫时间差异有统计学意义（P＜0.01）.结论 Alkyl-PEI2k/SPIO标记成人外周血源性EPCs的效率高,且对细胞活性无明显影响;高场强7.0T MR能够用于Alkyl-PEI2k/SPIO标记的EPCs体外成像.

Objective To assess the feasibility of labeling endothelial progenitor cells （EPCs） with N-Alkyl-polyethylenimine 2 kDa stabilized superparamagnetic iron oxide （Alkyl-PEI2k/SPIO） and imaging in vitro by a 7.0 T MR scanner.Methods EPCs were obtained from human peripheral blood by density gradient centrifugation and cultured adherently under given conditions.Alkyl-PEI2k/SPIO （7 μg/ml Fe） was used to label EPCs by incubation overnight,and the labeling efficiency was determined by Prussian blue staining.The viability and activity of labeled cells were evaluated using CCK-8,Transwell migration and tubulogenesis assays.Labeled EPCs agarose hydrogels with different number of cells （0,5×10^3,1×10^4,5×10^4,1×10^5,2.5×10^5,5×10^5,1×10^6 and 1.5×10^6） were prepared,and in vitro 7.0 T MR was performed.Signal intensity （SI） and relaxation time were measured.Data were analyzed by two-sample t test and paired t test.Results The labeling efficiency with Alkyl-PEI2k/SPIO at a 7 μg Fe/ml concentration was 100%.Quantitative analysis of cellular iron was （6.062±0.050） pg/cell.There was no statistical difference between the absorbance of labeled and unlabeled EPCs determined by CCK-8 assay （P〉0.05）.Migration assay showed that the number of labeled migrated EPCs （80.6±8.0） was comparable to that of unlabeled EPCs （77.6±4.6; P〉0.05）.Tubulogenesis assay suggested that the number of tube-like structures formed by labeled EPCs （9.0± 1.0） was not statistically different from that of the unlabeled EPCs （9.4± 1.5 ; P〉0.05）.In vitro MRI showed that SI and relaxation time were reduced with the increased number of labeled EPCs.Both SI and relaxation time had significant differences between T2 and T2＊ （P〈0.01）.Conclusions EPCs could be labeled with Alkyl-PEI2k/SPIO efficiently without significant side effect on cell activity.Magnetically labeled EPCs could be imaged in vitro by a 7.0 T MR scanner.