兔骨髓基质细胞Feridex标记及体外MRI成像的实验研究

Experimental study on Feridex labeling and in vitro MRI for rabbit bone marrow stromal cells

目的使用超顺磁性氧化铁菲立磁(FE)和转染试剂多聚赖氨酸(PLL)对骨髓基质细胞(BMSCs)进行体外标记,并在体外对标记细胞进行MRI示踪,确定其标记BMSCs的适宜浓度。方法使用不同浓度(10、25、50、75μg/mL)的FE复合PLL,形成FE-PLL并标记新西兰大白兔BMSCs后,分为5组(A:纯BMSCs组;B:5μg/mL FE+0.75μg/mL PLL组;C:12.5μg/mL FE+0.75μg/mL PLL组;D:25μg/mL FE+0.75μg/mL PLL组;E:37.5μg/mL FE+0.75μg/mL PLL组)。对各组细胞分别进行普鲁士蓝染色、流式细胞仪检测、透射电镜观察,检测FE-PLL标记兔BMSCs的效率及其对凋亡的影响。体外扫描采用4.7T MR机,扫描序列为轴面T2WI。结果FE可以高效率标记BMSCs,被标记的细胞显微镜下呈淡黄或深黄,颜色的深浅与所加FE的剂量呈正相关:胞质内散在分布许多含膜的囊泡样包涵体结构,囊泡内有浓聚细小的铁颗粒,从B组～E组依次增多。流式细胞仪结果显示,5、12.5、25μg/mL各组FE对细胞无不良影响,而37.5μg/mL组凋亡细胞明显增加。体外4.7T MRI显示未标记组无信号改变,呈显著高信号,标记组随FE浓度的增高信号改变增大,信号降低的越明显。结论FE可以用来体外标记兔BMSCs,在适宜浓度下对其生物学活性无明显影响,并能在MRI下达到示踪的目的。

Objective To label bone marrow stromal cells （BMSCs） with the superparamagnetic iron oxide Feridex （FE） and the transfection agent poly-1-lysine （PLL）, and track the labeled cells with MRI in vitro to determine the optimal concentration of FE to label BMSCs in vitro. Methods FE-PLL complexes with different concentrations of FE （10, 25, 50, 75 μg/mL） were used to label BMSCs of New Zealand rabbits, which were divided into five groups （A： pure BMSCs group; B： 5 μg/mL FE＋0.75 μg/mL PLL group; C： 12.5 μg/mL FE＋0.75 μg/mL PLL group; D： 25 μg/mL FE＋0.75 μg/mL PLL group; E： 37.5 μg/mL FE＋0.75 μg/mL PLL group）. The efficiency of FE-PLL labeling rabbit BMSCs and its impact on cellular apoptosis were evaluated by Prussian blue staining, flow cytometry and transmission electron microscopy. The 4.7T MR machine was applied for scanning in vitro, sequences of scan was axial T2WI. Results BMSCs could be effectively labeled with FE. The labeled cells were lightly or deeply yellow under the microscope, color of which positively correlated with the added FE dose. An abundance of membrane-containing vesicle-like inclusion bodies scattered in the cytoplast, accumulation of fine iron particles within the vesicles and the number of iron particles increasing gradually from group B to E. Flow cytometry assay demonstrated that FE in the experiment groups （5, 12.5 and 25 μg/mL） had no adverse effect on BMSCs except that cell apoptosis increased evidently in 37.5 μg/mL group. In vitro 4.7T MRI showed no signal change in unlabeled cells, but still with remarkably high signals; however, in labeled cell group, the more FE concentration increased, the more evidently signal intensity decreased. Conclusion FE can be applied to label rabbit BMSCs in vitro and FE with the optimal concentration had no effect on the biological activity of the labeled BMSCs; in addition, MRI can be used to track labeled cells in vitro.