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目的:探索超顺磁性氧化铁纳米颗粒(SPIO)标记猪骨髓间充质干细胞(MSCs)的合适方案。方法:分离培养猪MSCs,用不同SPIO浓度、不同共孵育时间进行标记,根据标记效率指标(普鲁士蓝染色、细胞铁浓度测定、细胞透射电镜检查)和标记毒性指标(台盼蓝排除试验、CCK-8试验)确定适宜的标记条件,然后对不同标记细胞进行磁共振(MR)体外成像。结果:(1)随着SPIO浓度的增加,MSCs细胞内平均含铁量相应升高;细胞涂片普鲁士蓝染色也显示细胞内蓝染颗粒相应增加。当SPIO浓度为25、50μg·mL-1时,细胞存活率和细胞活力与无SPIO时差异无统计学意义(P>0.05);但当SPIO浓度增加到100μg·mL-1时,CCK-8试验显示细胞活力下降(P<0.01);当SPIO浓度增加到200μg·mL-1时,细胞存活率和细胞活力均下降(P<0.05);(2)设定SPIO浓度为50μg.mL-1,随共孵育时间增加,细胞内含铁量逐渐升高(各组间比较均P<0.01);当标记时间为0、2、4、8、16、24h时,细胞存活率和细胞活力并差异无统计学意义(P>0.05);但当标记时间进一步延长到48h时,细胞存活率有所降低(P<0.05);(3)按“50μg/ml×24h方案”标记后,1.5TMR成像仪体外MRT2* WIflash检测阈值为7.5×104个细胞。结论:50μg·mL-1×24h方案是猪骨髓间充质干细胞比较合适的磁探针标记方案。
Objective: To explore the suitable solution of super-paramagnetic iron oxide nanoparticles (SPIO) labeled porcine bone marrow mesenchymal stem cells (MSCs). Methods: Porcine MSCs were isolated and cultured and labeled with different concentrations of SPIO and different incubation time. According to labeling efficiency indicators (Prussian blue staining, determination of iron concentration in cells, cell transmission electron microscopy) and labeling toxicity indicators (trypan blue exclusion test, CCK -8 test) to determine the appropriate labeling conditions, and then different labeled cells magnetic resonance (MR) in vitro imaging. Results: (1) With the increase of SPIO concentration, the average intracellular iron content of MSCs increased accordingly; Prussian blue staining of cell smears also showed a corresponding increase of intracellular blue dye particles. When SPIO concentration was 25 and 50μg · mL-1, the cell viability and cell viability were not significantly different from those without SPIO (P> 0.05). However, when SPIO concentration was increased to 100μg · mL-1, CCK-8 (P <0.01). The cell viability and cell viability decreased when the SPIO concentration was increased to 200 μg · mL-1 (P <0.05). (2) The SPIO concentration was set at 50 μg.mL-1 , With the increase of incubation time, the intracellular iron content increased gradually (all P <0.01); when the labeling time was 0, 2, 4, 8, 16 and 24 h, the cell viability and cell viability (P> 0.05). However, when the labeling time was further extended to 48h, the cell survival rate was decreased (P <0.05). (3) After labeling with “50μg / ml × 24h program” 1.5TMR imager in vitro MRT2 * WIflash detection threshold of 7.5 × 104 cells. Conclusion: The 50μg · mL-1 × 24h protocol is a suitable magnetic probe labeling protocol for porcine bone marrow mesenchymal stem cells.