外周血内皮祖细胞磁探针标记及体外磁共振成像的实验研究

目的应用纳米磁探针三氧化二铁（Fe2O3）-多聚赖氨酸（PLL）复合物体外标记兔外周血内皮祖细胞（EPCs）,磁共振（MR）对标记细胞成像。方法合成Fe2O3-PLL复合物。分离兔外周血单个核细胞,贴壁法筛选出EPCs,传代培养,Fe2O3-PLL标记祖细胞,普鲁士蓝染色、电子显微镜显示细胞内铁,四氮噻唑蓝（MTT）比色试验评价不同浓度Fe2O3-PLL标记EPCs后对细胞生长状况的影响,流式细胞分析检测标记、未标记细胞的细胞周期、细胞凋亡,应用MR的不同序列进行细胞群成像。结果普鲁士蓝染色、电镜观察均可见铁颗粒位于细胞质内,标记率接近100％,MTT比色试验示10μg／ml至200μg／ml7个铁浓度组,Fe2O3-PLL标记后细胞的光吸收值与未标记者比较,差异无统计学意义,流式细胞分析结果显示Fe2O3-PLL标记后细胞周期、细胞凋亡与未标记细胞间差异无统计学意义。磁共振成像（MRI）显示标记Fe2O3-PLL的细胞群较未标记者信号降低,以T2^＋加权像（FWI）信号强度变化率最大;标记后7d的信号强度变化率均较标记1d者下降。结论Fe2O3-PLL可以有效标记兔外周血EPCs,其对细胞的活力、增殖等生物学特性无明显影响。临床应用型1．5TMR可在体外进行标记细胞群成像,间接反映于细胞的数量和分裂增殖状态。

In vitro MR imaging of Fe(2)O(3)-PLL labelled rabbit peripheral blood endothelial progenitor cells

OBJECTIVE: To perform in vitro magnetic resonance imaging on magnetic iron oxide (Fe(2)O(3)-PLL) labeled rabbit peripheral blood endothelial progenitor cells (EPCs). METHODS: Fe(2)O(3) was incubated with PLL for 2 hours to form Fe(2)O(3)-PLL. Rabbit peripheral blood mononuclear cells (MNCs) were isolated and EPCs were selected by adherence method, expanded and incubated with Fe(2)O(3)-PLL. Intracellular iron was detected by Prussian blue stain and under electron microscope. MTT assay was used to evaluate cell survival and proliferation of Fe(2)O(3)-PLL labeled EPCs. Flow cytometry was used to analysis cell cycle and apoptosis. The cells underwent in vitro MR imaging with various sequences. RESULTS: Iron-containing intracytoplasmatic vesicles could be observed clearly with Prussian blue staining and electron microscope observation. Survival, life cycle and apoptosis values obtained by MTT and flow cytometry analysis were similar among unlabelled EPCs and EPCs labeled with various concentrations Fe(2)O(3)-PLL. The signal intensity on MRI was significantly decreased in labeled cells compared with that in unlabeled cells. The percentage change in signal intensity (DeltaSI) was most significant on T(2)*WI and DeltaSI was significantly lower in cells labeled for 7 days than that labeled for 1 day. CONCLUSIONS: The rabbit peripheral blood EPCs can be labeled with Fe(2)O(3)-PLL without significant change in viability and proliferation. The labeled EPCs can be imaged with standard 1.5 T MR equipment. The degree of MR signal decreasing may indirectly reflect the cells count, growth state and division.