纳米磁性探针转染大鼠外周血内皮祖细胞的磁共振成像实验研究

Magnetic resonance imaging of endothelial progenitor cells from rat peripheral blood labeled with nano-bioprobe of superparamagnetic iron oxide

目的建立稳定的体外分离培养、诱导分化大鼠外周血内皮祖细胞(endothelial progenitor cells,EPCs),及应用纳米磁探针体外标记EPCs的方法,磁共振(MR)进行标记细胞成像。方法抽取SD大鼠外周血,应用Ficoll密度梯度离心的方法获得单个核细胞,同时应用VEGF和bFGF诱导,使之向内皮细胞分化,以免疫细胞化学鉴定贴壁细胞的内皮标志。制备Fe2O3-arginine复合物,对EPCs进行磁探针标记,应用MR进行细胞群成像。结果经VEGF和bFGF诱导后的EPCs的内皮标志CD31、CD34、Flk21和vWF在不同时段呈阳性表达。普鲁士蓝染色可见铁颗粒位于细胞质内,标记率接近100%;磁共振成像(MRI)显示标记的细胞群信号强度的变化较未标记细胞群信号降低。结论大鼠外周血中含有EPCs,在体外特殊诱导环境下,可定向分化为内皮样细胞。Fe2O3-arginine可以有效标记EPCs构建纳米生物探针,临床应用型1.5TMR可在体外进行标记细胞群成像。

Objective To investigate the isolation, culture, proliferation and differentiation of endothelial progenitor cells （EPCs） from rat circulating blood and to study of magnetic resonance imaging of nano-bioprobe that was constructed with endothelial progenitor cells and superparamagnetic iron oxide （SPIO）. Methods The mononuclear cells （MNCs） were isolated from SD rat peripheral blood, and EPCs were selected by adherence method. Then the expressions of cell markers of EPCs were assessed by immunohistochemistry. EPCs were incubated with F2O3-arginine for 24 h, then intracellular iron was detected by Prussian blue stain and the cells underwent in vitro MR imaging with three sequences （TIWI, T2WI, T2 ＊ WI）. Results Immunocytochemical stain showed that the adherent cells were positive for CD31, CD34, Flk21 and vWF 4 d after adherent culture. Iron-containing intracytoplasmatic vesicles could be observed clearly with Prussian blue staining, and the labeling rate of EPCs were nearly 100%. The signal intensity on MRI was significantly decreased in labeled EPCs compared with unlabeled cells. Conclusion EPCs can be obtained from mononuclear cells in rat peripheral blood and be differentiated into endothelial cells. EPCs can be labeled with Fe2O3-arginine to construct nanobioprobe in vitro without significant changes in viability and proliferation. The labeled EPCs can be imaged with standard 1.5 T MR equipment.