猪骨髓间充质干细胞超顺磁性氧化铁标记方法的实验研究

The Experimental Study of Superparamagnetic Iron Oxides-Labeled Swine Bone Marrow Mesenchymal Stem Cells in vitro and the Effect on Magnetic Resonance Imaging Signals

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 optimal labeling procedure on swine bone marrow mesenchymal stem cells（MSCs） with superparamagnetic iron oxides（SPIO） in vitro and its effect on magnetic resonance（MR） iminging signals. Methods： MSCs isola ted from swine were incubated with SPIO nanoparticles on different concentrations and times. The location, distribution and the labeling ratio of SPIO particles in MSCs was assessed by Prussian Blue staining,electron microscopy and atomic absorption spectrometry. Cell viability was observed by trypan-blue exclusion and CCK-8 test. At an optimal concentration, samples of SPIO-labeled MSCs suspension in agar were imaged by MR imaging with T2 ＊ WI-Flash. Results： （1） With the increase of SPIO concentration, the number of intracytoplasmic particles stained with Prussian Blue stain and the mean cellular iron content increased accordingly. When SPIO concentration reached 100μg· mL^-1, CCK-8 test showed remarkable decrease of labeled cells viability （P〈0.01）, while there was no significant difference among those with SPIO concentration of 0μg· mL^-1 ,25 μg· mL^-1 and 50 μg· mL^-1 （P〈0.05）. When SPIO concentration went up to 200 μg· mL^-1,the survival rate and viability of the labeled ceils both decreased significantly （P〈0.05）. （2） As the incubation time extended, the mean cellular iron content at different time points increased gradually under fixed SPIO concentration of 50 μg· mL^-1. The survival rate and viability of the labeled cells showed no significant differences among groups incubated for 0,2,4,8, 16, 24 h, respectively, while decreased tremendously at 48 h （P〈0.05）. （3）As few as 7. 5 × 10^4 cells could be detected by a standard 1.5 T MR imaging with T2· WI-Flash scanning sequence using labeling procedure of 50 μg · mU^-1× 24 h. Conclusion：The labeling procedure of 50 μg · mL^-1×24 h may be an optimal procedure for magnetic labeling of swine bone marrow stem cells. Key Words Superparamagnetic iron oxides; Mesenchymal stem ceils; Labeling