超顺磁性氧化铁标记胚胎干细胞的磁共振成像

Wang Bei, Jaconi Marisa, Li Jian, Wang Yan, Valle Jean-Paul

Department of Emergency, Beijing Hospital, Beijing 100730, China.

Zhonghua Yi Xue Za Zhi. 2007 Jun 19;87(23):1646-8.

OBJECTIVE

To evaluate the labeling efficiency of superparamagnetic iron oxide (SPIO) nanoparticles and its toxicity to mouse embryonic stem cells (ESCs) and (embryoid body (ES)-derived cardiomyocytes.

METHODS

Mouse ESCs of the line CGR8 were cultured and induced to differentiate into ES-derived cardiomyocytes. The EB-derived cardiomyocytes were coincubated with SPIO contrast agent at different concentrations (1, 8, 9.3, 14, 28, and 56 mg/L) and transfection agent for 24 and 48 hours for cell labeling. Cells not labeled by SPIO and cells labeled by SPIO without transfection agent were used as controls. Spectrophotometer was used to detect the iron concentration in the cells. Confocal microscopy was used to test the intracellular calcium levels ([Ca(2+)] i). The ultrastructure of the cells was observed by electron microscopy. Ex vivo MRI was used to observe the signals of the cells.

RESULTS

Iron-containing intracytoplasmic vesicles could be observed clearly with electron microscopy. The intracellular iron concentration was higher in the cells treated with transfection agent than in the cells not treated with transfection agent. The iron concentration of the cells treated with the SPIO at the concentration of 9.3 microg/ml for 24 hours was the highest. There were no differences in the morphology, contractile areas (chi(2) = 1.32; P = 0.25), and the beating frequency (t = 1.73; P = 0.10) between the EBs from iron-labeled ESCs and from the control ESCs. The rhythmic intracellular free Ca(2+) fluctuation in the labeled cardiomyocytes was similar to that of the controls. The MR images with T(2)WI and T(2)WI sequences, especially those with T(2)WI sequence, of the ESCs showed that the signals of the SPIO labeled cells were lower than those of the SPIO-labeled cells.

CONCLUSION

SPIO labeling of ESCs and ES-derived cardiomyocytes does not influence the cell viability and proliferation. The standard 1.5T MR equipment can image the labeled cells, thus offering the possibility of cell tracking and migration monitoring in MRI.

目的

评估超顺磁性氧化铁（SPIO）纳米颗粒的标记效率及其对小鼠胚胎干细胞（ESCs）和胚状体（EB）来源的心肌细胞的毒性。

方法

培养CGR8系小鼠胚胎干细胞并诱导其分化为EB来源的心肌细胞。将EB来源的心肌细胞与不同浓度（1、8、9.3、14、28和56 mg/L）的SPIO造影剂和转染剂共孵育24和48小时进行细胞标记。未用SPIO标记的细胞和用SPIO但未用转染剂标记的细胞用作对照。用分光光度计检测细胞中的铁浓度。用共聚焦显微镜检测细胞内钙水平（[Ca(2+)]i）。通过电子显微镜观察细胞的超微结构。用离体磁共振成像观察细胞的信号。

结果

电子显微镜下可清晰观察到含铁的胞质内小泡。用转染剂处理的细胞内铁浓度高于未用转染剂处理的细胞。用浓度为9.3μg/ml的SPIO处理24小时的细胞铁浓度最高。铁标记的胚胎干细胞来源的EB与对照胚胎干细胞来源的EB在形态、收缩面积（χ(2)=1.32；P = 0.25）和搏动频率（t = 1.73；P = 0.10）方面无差异。标记的心肌细胞中细胞内游离钙的节律性波动与对照相似。胚胎干细胞的T(2)WI和T(2)WI序列的磁共振图像，尤其是T(2)WI序列的图像显示，SPIO标记细胞的信号低于未标记细胞的信号。