不同种类超顺磁性氧化铁标记猪骨髓间充质干细胞的体外MR成像

背景:干细胞磁性标记是新近开展的一项干细胞体外标记技术,结合MR成像设备可以活体监控移植入体内的干细胞.目的:明确超顺磁性氧化铁粒子体外标记猪骨髓间充质干细胞的方法、不同种类超顺磁性氧化铁标记细胞经MR成像的特征及可成像的最低标记细胞量.设计、时间及地点:对比观察,于2006-09/2007-03在苏州大学医学部心血管外科实验室及苏州大学附属第一医院影像中心完成.材料:猪髂骨骨髓由太湖梅山猪新鲜采集:超顺磁性氧化铁纳米颗粒为德国Schering公司产品;超微型超顺磁性氧化铁纳米颗粒由苏州大学化学与化工学院提供:铁颗粒晶核表面包被葡聚糖,3种超微型超顺磁性氧化铁包被葡聚糖后根据颗粒大小(12,15,20 nm)分别依次简称为1#,2#,3#.方法:分离、纯化、培养猪骨髓间充质干细胞,体外进行不同种类超顺磁性氧化铁标记,染色及荧光显微镜观察;测量并绘制未标记细胞和标记细胞的MTT生长曲线;选取不同的细胞量组(Feridex标记细胞分别选取1×106、5×105及1×105L-1量组,未标记细胞选取5×105L-1量组,1#、2#及3#超微型超顺磁性氧化铁标记细胞均选取5×105L-1量组)进行标记后MR成像,测量不同扫描序列标记细胞管的信号强度改变,并进行统计学分析.主要观察指标:超顺磁性氧化铁标记干细胞的普鲁士蓝染色检测标记率:标记干细胞的MTT生长曲线;双染色法检测细胞凋亡;不同Ependoff管内细胞团T1WI、T2WI和FFE图像的信号强度.结果:应用多聚赖氨酸介导干细胞磁标记方法标记骨髓间充质干细胞有效率为100%,普鲁士蓝染色见细胞浆内有多少不等的蓝染铁颗粒;超顺磁性氧化铁标记的间充质干细胞在T2WI尤其是FFE(T2WI)序列信号明显降低;在25 mg/L Fe培养液标记浓度下,MR成像的最低细胞量为1×105;在不同种类超顺磁性氧化铁标记下,2#、3#USPIO与Feridex在T2WI及T2*WI上有显著性差异(P<0.01);而1#USPIO与Feridex在T2WI及T2WI上差异无显著性意义(P>0.05):Feridex标记间充质干细胞在T2WI及T2WI上与T1WI相比,差异均有显著性意义(P<0.01).结论:超顺磁性氧化铁可以简便标记间充质干细胞并且在适当浓度下对间充质干细胞的生物学活性没有影响,MR T2WI和T2*WI序列可敏感显像磁性标记的干细胞.

In vitro magnetic resonance imaging of porcine bone marrow mesenchymal stem cells labeled by various kinds of superparamagnetic iron oxide nanoparticles

BACKGROUND: Magnetic labeling of stem cells is a recently developed stem cell in vitro labeling technique. Through in conjunction with magnetic resonance imaging (MRI), it can monitor transplanted stem cells in vivo. OBJECTIVE: To identify the method of superparamagnetic iron oxide (SPIO) labeling pig bone marrow mesenchymal stem cells (BMSCs), to investigate the characteristics of stem cells labeled by various SPIO following MRI, and to determine the minimum amount of labeled cells fer MRI. DESIGN, TIME AND SETTING: A control observation was performed at the laboratories of Department of Cardiovascular Surgery, Medical College of Soochow University, and Medical Imaging Centre, First Affiliated Hospital, Soochow University between September 2006 and March 2007. MATERIALS: Fresh porcine iliac bone marrow was collected from Taihu Meishan pigs. SPIO nanometer particles were purchased from Schedng, Germany. Ultramicro SPIO (USPIO) nanoparticles were provided by School of Chemistry and Chemical Engineering, Soochow University. For such particles, crystal nucleus surface was coated with dextran, and following coating, they were named 1#, 2#, 3# for short according to particle size. METHODS: Following isolation, purification, and culture, BMSCs were in vitro labeled by various kinds of SPIO nanoparticles. The labeled cells were subjected to Prussian blue staining and fluorescence microscope observation. The cell growth was observed using MTT method and the growth curve was plotted. For Feridex-labeled cells, 1×106), 5×105, and 1×105 three cell amount groups were set, for unlabeled cells, a 5×105 cell amount group was included, and for 1#, 2#, and 3# SPIO-labeled cells, only 5×105 cell amount group was used. MRI was conduced for measurement of signal intensity of cells labeled by different scanning sequences, followed by statistical analysis. MAIN OUTCOME MEASURES: Detection of SPIO nandparticles-labeled cells by Prussian blue staining; Growth curves of SPIO nanoparticles-labeled cells; Detection of cellular apoptosis by double staining; Determination of signal intensity of cell masses from different Ependoff tubes using MRI with T1WI, T2WI, and fast field echo (FFE) sequences. RESULTS: BMSCs could be labeled with SPIO and the labeling efficiency was 100%. Different amounts of blue-stained Fe particles could be observed in the cytoplasm by Prussian blue staining. SPIO labeling caused a significantly lower signal attenuation effect in T2WI and FFE (T2*WI) images than in T1WI images. In a labeling concentration of 25 mg/L Fe solution, the minimum cell amount for MRI was 1 x 105. The signal intensity exhibited significant difference in 2#, 3#USPIO- and Feridex-labeled cells in no matter T2WI or T2*Wl images(P < 0.01). But no significant difference was found between 1#USPIO- and Feridex-labeled cellss in no mater T2WI or T2*WI images(P > 0.05). There was significant difference in signal intensity of Feridex-labeled BMSCs between T2WI, T2*WI and T1Wl images (P < 0.01). CONCLUSION: BMSCs can be easily and efficiently labeled by SPIO nanoparticles without interference, at proper concentrations on cell viability and proliferation. MRI visualization of SPIO-labeled BMSCs is feasible in both T2WI and T2*WI images.