新型超顺磁性氧化铁标记血管内皮祖细胞的实验研究

目的 研究新型超顺磁性氧化铁(SPIO)标记骨髓源血管内皮祖细胞(EPCs)对其生物学影响,提高其标记效率及安全性.方法 采用梯度离心结合贴壁法分离纯化SD大鼠骨髓源EPCs并鉴定,以3-氨丙基三乙氧基硅烷(APTS)修饰Fe2O3配制成SPIO,用六种不同浓度SPIO(0 μg/ml、6.25 μg/ml、12.5 μg/ml、25 μg/ml、50 μg/ml、100 μg/ml)分别转染标记EPCs,对标记后的EPCs行普鲁士蓝染色、台盼蓝染色、透射电镜、噻唑蓝(MTT)检测、流式细胞仪检测细胞凋亡与细胞周期,探讨不同SPIO浓度对EPCs标记效率、细胞活性及增殖的影响.结果 普鲁士蓝染色显示EPCs呈浓度依赖性摄取SPIO,培养基中Fe3+浓度为25 μg/ml时,标记效率最高,可见95％以上EPCs普鲁士蓝染色阳性;当Fe3+浓度小于12.5 μg/ml时,染色阳性细胞数小于50％;当Fe3+浓度为100μg/ml时活细胞数目小于40％,与对照组比较差异有统计学意义(P<0.05).台盼蓝染色显示随着标记浓度的升高,细胞活力逐渐升高.当SPIO浓度超过50 μg/ml,细胞活性逐渐降低.透射电镜可见EPCs超微结构保存良好,大量的高密度SPIO颗粒,主要位于溶酶体内、滑面内质网、线粒体等细胞器的膜结构上.MTT测试及细胞凋亡与周期检测表明SPIO标记对EPCs存活、增殖的能力无明显影响,标记后的EPCs可用于进一步干细胞示踪研究.结论 梯密度离心结合贴壁法可分离出血管内皮祖细胞进行体外增殖分化.APTS修饰Fe2O3配制而成的新型SPIO可简便标记EPCs,并且在适当浓度下对MSCs的生物学活性没有影响,为后期的活体MRI示踪于细胞移植实验奠定基础.

Study on endothelial progenitor cells labeling with new superpara-magnetic iron oxide

Objective To research the biological effects of labeling endothelial progenitor cells(EPCs) with new superpara-magnetic iron oxide (SPIO), and improve its efficacy and safety. Methods EPCs were extracted and purified from rat bone marrow by density-gradient centrifugation and the method of adherence-sieve, and then incubated with six different concentrations of SPIO particles (0 μg/ml, 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml), which were made up with Fe2O3 embellished by APTS. We explored the labeling efficiency and the effects on cell proliferation and cell activity of SPIO by using Prussian blue staining, transmission electron microscopy, MTT test and flow cytometry. Results Prussian blue staining showed that SPIO was taken in by EPCs in a concentration dependence manner. There was the highest labeling efficiency about 95% Prussian blue staining positive cell when the concentration of Fe3+ was 25 μg/ml, 50% when the concentration of Fe3+ was under 12.5 μg/ml, and the number of living cell was under 40% when the concentration of Fe3+ was 100 μg/ml, showing a significant difference(P<0.05). Transmission electron microscopy showed the ultrastructure of EPCs was well preserved. Abundant SPIO granules were found in organelles, such as lysosome, smooth endoplasmic reticulum and mitochondrion. MTT test and flow cytometry showed that cell viability, proliferation and differentiation ability of labeled cells were not affected, and the labeled cell can be used stem cell tracer in vivo. Conclusion Bone marrow-derived EPCs can be successfully isolated and cultured by density-gradient centrifugation and adherence-sieve, and labeled by SPIO easily and efficiently without interference on the cell viability and proliferation, which lay the foundations for the further stem cell tracer in vivo.