超顺磁性氧化铁标记大鼠骨髓间充质干细胞的研究

目的探讨应用超顺磁性氧化铁（superparamagnetic iron oxid,SPIO）标记间充质干细胞（mesenchymal stemcells,MSCs）的有效性、安全性及可靠性。方法分离、培养SD大鼠MSCs,通过流式细胞仪分析鉴定表面抗原CD34、CD44。选取第4代MSCs,在铁（Fe）浓度为25、50、75μg.ml-1和多聚赖氨酸（Poly-L-Lysine,PLL,0.75μg.ml-1）的DMEM/F12培养液中,孵育48h,作为3个实验组。通过普鲁士蓝染色、流式细胞仪检测、台盼蓝染色及MTT细胞增殖活性检测SPIO,探讨不同SPIO浓度对MSCs标记效率、免疫学表型、细胞活性及增殖的影响。结果经Fe浓度为25、50、75μg.ml-1SPIO标记后,普鲁士蓝染色标记效率均在99%以上;3个实验组细胞表面均表达CD44,阳性细胞率为超过98%。与对照组相比,Fe浓度为25、50μg.ml-1的SPIO标记的MSCs活细胞比率均在96%左右,细胞增殖活性差异无统计学意义,而Fe浓度为75μg.ml-1时,活细胞比率约为93%,对细胞的增殖有明显的抑制作用（P〈0.05）。培养4周,细胞内检测不到SPIO。结论全骨髓直接贴壁法可以成功分离、培养MSCs。超顺磁性氧化铁在PLL介导下,能高效地标记MSC,且在合适的浓度范围不会影响MSCs的免疫学表型、细胞活性及增殖能力等生物学特性。细胞内的SPIO,大约在4周左右,即可被完全降解。

Studies on rat mesenchymal stem cells labeling with superparamagnetic iron oxide

Objective To investigate the efficacy,safety and reliability of labeling mesenchymal stem cells（MSCs） with superparamagnetic iron oxid（SPIO）.Methods MSCs were isolated and cultured from 4-6 weeks old rats bone marrow by plastic adherence method.MSCs were identified by specific surface antigens CD34 and CD4 by flow cytometry.The MSCs of the fourth passage were incubated with superparamagnetic iron oxide and poly-L-Lysine（SPIO-PLL） complexes containing concentrations of 25,50 or 75μg iron and 0.75μg poly-L-Lysine per milliliter for 48 hours.Labeled cells were compared with unlabeled cells by Prussian blue staining,flow cytometry,Trypan blue staining and MTT test to evaluate the effects of SPIO on cell labeling efficiency,surface markers expressions,cell viability and cell proliferation.Results After incubation with SPIO,the labeling efficiency was about 99%.Results also showed that similar CD44 expression frequency（98%） was obtained between labeled and unlabeled cells.Cell viability in cells labeled with SPIO-PLL at concentrations of 25 or 50 μg iron per milliliter together with 0.75μg PLL per milliliter were similar to that of the unlabeled cells.While labeling at concentration of 75μg iron per milliliter,the cell viability rate was decreased and cell proliferation was significantly inhibited（P0.05）.After 4 weeks in tissue culture,no SPIO was detactable in cells.Conclusion Using the whole bone marrow plastic adherence method,rat MSCs can be successfully isolated and cultured.SPIO-PLL can efficiently label MSCs safely and effectively at optimal concentrations of SPIO（25 to 50μg iron together with 0.75μg PLL per milliliter）.Intracellular SPIO was completely degraded in 4 weeks.