磁性标记胎盘间充质干细胞的最佳浓度筛选

Screening of Optimal Concentration of Magnetic Labeled Placental Mesenchymal Stem Cells

目的探讨超微超顺磁性氧化铁(USPIO)标记人胎盘间充质干细胞(hPMSCs)的最佳浓度区间,为实现其磁共振(MR)体内动态成像和临床应用提供基础研究资料。方法分离培养hPMSCs,以多聚赖氨酸(PLL)为转染剂,与不同浓度USPIO形成PLL-USPIO复合物,分别标记hPMSCs并考察其标记效率,检测标记后h PMSCs的存活率、增殖能力和凋亡率等生物学特性。结果普鲁士蓝染色均显示细胞质内大量铁颗粒存在,当标记浓度≥10μg·mL^-1时标记率均>95%;当磁性标记浓度为15、20和25μg·mL^-1时hPMSCs存活率均低于对照组(P均<0.01);标记浓度在25μg·mL^-1内时增殖能力与对照组比较在第1天、3天、5天差异均无统计学意义(P均>0.05),培养至第7天标记浓度为25μg·mL^-1组的OD值低于对照组(P=0.047);各标记细胞与未标记细胞晚期凋亡率差异均无统计学意义(P均>0.05),早期凋亡率随标记浓度增加呈递增趋势,标记浓度≥15μg·mL^-1时早期凋亡率高于对照组(P<0.05),在标记浓度为20、25μg·mL^-1总凋亡率高于对照组(P<0.05)。结论USPIO浓度区间为5~15μg·mL^-1时可以对hPMSCs进行有效标记且对其活性、增殖能力和凋亡率等生物学特性无明显影响。

Objective To investigate the optimal concentration range of ultramicro superparamagnetic iron oxide(USPIO)labeled human placental mesenchymal stem cells(hPMSCs),and provide basic research data for real-time MR imaging and clinical application.Methods hPMSCs were isolated and cultured,and poly-lysine(PLL)was used as transfection reagent to form PLL-USPIO complex with different concentrations of USPIO to label hPMSCs and investigate the labeling efficiency.Biological characteristics of labeled hPMSCs were detected:cell survival rate was calculated by trypan blue exclusion test,cell proliferation was detected by cell count(CCK-8)kit,and flow cytometry was used to judge the apoptosis of the cells.The difference of cell proliferation ability was analyzed by Kruskal Wallis testing,and the cell survival rate and apoptosis rate were compared by one way ANOVA.Results Prussian blue staining showed that a large number of iron particles existed in the cytoplasm,the labeling rate was more than 95%when the labeling concentration was more than10μg·m L^-1.When the final magnetic labeling concentration was above 15μg·m L^-1,the cell survival rate began to decrease significantly,and the hPMSCs survival rates were lower than those of the control group at labeling concentrations of 15,20 and 25μg·m L^-1(P all<0.01).There was no difference in the proliferation ability between the labeled concentration within 25μg·m L^-1 and the control group on the 1 st,3 rd and 5 th(P all>0.05),but the OD value of 25μg·m L^-1 group was significantly lower than that of control group on the 7 th day(P=0.047).There was no significant difference in the late apoptotic rate between labeled cells and unlabeled cells(P all>0.05),but the early apoptotic rate showed an increasing trend with increasing labeling concentration,and the early apoptotic rate at labeling concentration above 15μg·m L^-1 was higher than the control group(P<0.05),the total apoptotic rate at the labeled concentration of 20,25μg·m L^-1 was higher than the control group(P<0.05).Conclusion When the concentration range of USPIO is between 5 to 15μg·m L^-1,hPMSCs can be effectively labeled and has no significant effect on its activity,proliferation and apoptosis rate.