摘要
目的初步探索利用菲立磁(FE)和转染试剂多聚赖氨酸(PLL)体外磁性标记兔骨髓基质细胞(BMSC)这一方法的可行性,并确定其标记BMSC的最佳浓度。方法无菌条件下行股骨取髓,采用梯度密度离心法分离获取兔BMSC,体外培养、诱导分化为神经干细胞(NSC),使用不同浓度(5、12.5、25、37.5μg/ml)的FE-PLL标记BMSC。采用普鲁士蓝染色、CCK-8法、流式细胞仪、透射电镜、免疫细胞染色等方法,检测FE-PLL标记兔BMSC的效率及其对细胞生长、分化、凋亡的影响。结果FE可以高效率标记BMSC,被标记的细胞在显微镜下呈淡黄或深黄,颜色深浅与所加FE的剂量呈正相关。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。CCK-8测定的生长曲线表明:除37.5μg/ml组外,其他各组细胞之间差异无统计学意义。普鲁士蓝染色显示:各组FE-PLL标记BMSC胞质内均可见到细小的蓝色铁颗粒。电镜结果显示:各组FE-PLL标记的BMSC胞质内含有许多包裹铁颗粒的囊泡,其含铁囊泡的数量依FE浓度增加而增加。结论FE可以用来体外标记兔BMSC;其标记效率随FE浓度增加而增高,最佳浓度为25μg/ml。
Objective To explore the feasibility of protocols using Feridex (FE) and transfection agent, poly-l-lysine (PLL), for magnetic labeling of bone marrow stromal cells (BMSCs), and determine the optimal concentration of FE for labeling BMSCs in vitro. Methods Under sterile condition, the rabbit BMSCs were harvested by density gradient centrifugation following a thighbone puncture, and cultured in vitro and induced to differentiate into neural stem cells. Different concentrations (5, 12.5, 25, 37.5μg/ml) of FE-PLL complexes were used to magnetically label BMSCs. The efficiency and cellular viability of FE-PLL-labeled BMSCs were evaluated by Prussian blue staining, CCK-8 method, flow cytometry, transmission electron microscopy and immunocytochemical staining. The proliferation, differentiation and apoptosis of FE-PLL-labeled BMSCs were also investigated. Results BMSCs could be effectively labeled by FE. The labeled cells appeared light-yellow or deep-yellow under microscope, which was dependent on the dose of FE. Flow cytometry assay demonstrated that cell apoptosis was not increased significantly in the experimental groups (5, 12.5, 25μg/ml) compared with the control group. However, cell apoptosis was increased significantly in 37.5μg/ml group. Cell growth curves by CCK-8 assay showed that there were no significant differences between different groups (except 37.5μg/ml group). Prussian blue staining showed numerous fine blue iron particles in the cytoplasm of FE-PLL-labeled BMSCs. Transmission electron microscopy revealed the presence of numerous vesicles filled with electron-dense magnetic iron particles in the FE-PLL-labeled BMSCs. Endosomes filled with iron particles grew in number on the dose of FE. Conclusion FE can be used to label BMSCs in vitro, and the labeling efficiency is improved by increasing doses of FE. The optimal labeling concentration is 25μg/ml.
出处
《中国微侵袭神经外科杂志》
CAS
2007年第3期125-128,共4页
Chinese Journal of Minimally Invasive Neurosurgery
基金
国家自然科学基金(30400464)
广东省自然科学基金(04300199
05004763)
广东省名医工程研究项目(粤卫[2004]199号)
中国科学院武汉物理和数学研究所波谱与原子分子物理实验室基金(T152606)资助
关键词
骨髓基质细胞
神经干细胞
电子自旋共振谱学
聚赖氨酸
生物学标记
bone marrow stromal cells
neural stem cells
electron spin-resonance spectroscopy
polylysine
biological markers