超顺磁性氧化铁标记骨髓间充质干细胞的实验研究

Experimental study on superparamagnetic iron oxide labeled bone marrow mesenchymal stem cells

目的探讨超顺磁性氧化铁纳米颗粒(SPIO)标记青山羊骨髓间充质干细胞的方法,并确定标记颗粒在细胞内的具体位置,评价标记效果。方法抽取骨髓离心,分离间充质干细胞制成单细胞悬液传代培养及扩增,用SPIO标记第三代骨髓间充质干细胞,显微镜下观察发现细胞内胞吞入大量SPIO颗粒后消化培养细胞,离心成团,于2.5%戊二醛固定,透射电镜常规制样,镜下观察并采集图像。结果电镜下骨髓间充质干细胞的超微结构保存良好,胞质内出现大量电子密度高的SPIO颗粒,呈小团状或点状分布,主要位于溶酶体内及滑面内质网、线粒体及胞浆中的膜性结构上,细胞膜表面和核膜上也可见少量的散在颗粒。结论透射电镜下显示用SPIO标记骨髓间充质干细胞阳性率高,超微结构保存良好,准确定位了SPIO在胞内的位置,此法可为干细胞移植的示踪提供依据。

Objective To explore the methods of labeling bone marrow mesenchymal stem cells（BMMSCs） with superparamagnetic iron oxides（SPIO） as well as to confirm the location of SPIO in BMMSCs and to evaluate the labeling effect.Methods BMMSCs were isolated from grey goat and purified for subsequent steps.SPIO particles and the third generation of BMMSCs were cultured together till lots of SPIO particles were founded in the cell through microscope.The cells were digested and centrifuged to be a pellet and then were fixed by 2.5% pentadialdehyde.Samples were prepared for transmission electron microscope（TEM） examination and TEM pictures were captured for analysis.Results The ultrastructure of BMMSCs was well preserved.Abundant granules were found in cytoplasm,such as lysosome,smooth endoplasmic reticulum and mitochondrion.Small amount of interspersed granules were found on cell membrane and nuclear membrane.Conclusion TCM observation showed that the rate of positive cell that labeled by SPIO was high,the ultrastructure was well perserved and the location of SPIO was confirmed in the cell.This technique laid a foundation for the tracing of the stem cell transplantation.