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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(12,15,20nm)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(T2*WI)序列信号明显降低;在25mg/LFe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T2*WI上差异无显著性意义(P>0.05);Feridex标记间充质干细胞在T2WI及T2*WI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,MRT2WI和T2*WI序列可敏感显像磁性标记的干细胞。
BACKGROUND: Stem cell magnetic labeling is a newly introduced in vitro technique for stem cell labeling. In combination with MR imaging equipment, stem cells transplanted into the body can be monitored in vivo. OBJECTIVE: To identify the method of in vitro labeling of bone marrow mesenchymal stem cells with superparamagnetic iron oxide particles, the characteristics of MR imaging of different types of superparamagnetic iron oxide labeled cells, and the minimum labeled cell number that can be imaged. DESIGN, TIME AND SETTING: The comparative observation was performed at the Cardiovascular Surgery Laboratory, Medical College of Soochow University and the Imaging Center, First Affiliated Hospital, Soochow University from September 2006 to March 2007. Materials: Porcine iliac bone marrow was freshly collected from Meishan pig in Taihu Lake; superparamagnetic iron oxide nanoparticles were the products of Schering Company of Germany; ultramicro superparamagnetic iron oxide nanoparticles were provided by the School of Chemistry and Chemical Engineering of Soochow University: iron particle nucleus surface package Dextran, three kinds of ultra-superparamagnetic iron oxide coating dextran according to the size of the particles (12,15,20 nm) were followed by the abbreviation 1 #, 2 #, 3 #. Methods: Bone marrow-derived mesenchymal stem cells were isolated, purified, and cultured. Different types of superparamagnetic iron oxide labeled, stained and observed by fluorescence microscopy were observed in vitro. MTT growth curves of unlabeled cells and labeled cells were measured and plotted. Different cell numbers (Feridex labeled cells were selected 1 × 106,5 × 105 and 1 × 105L-1 volume group, unlabeled cells selected 5 × 105L-1 volume group, 1 #, 2 # and 3 # super-miniature superparamagnetic iron oxide Labeled cells were selected 5 × 105L-1 volume group) were labeled MR imaging, measurement of different scanning sequence marked cell tube signal intensity changes, and statistical analysis. MAIN OUTCOME MEASURES: Prussian blue staining detection rate of superparamagnetic iron oxide labeled stem cells; MTT growth curve of labeled stem cells; Dual-staining assay of apoptosis; Signal intensity of T1WI, T2WI and FFE images in different Ependoff tube groups. Results: The efficiency of labeling MSCs with polylysine-mediated magnetic labeling of stem cells was 100%. Prussian blue staining showed that there were many blue-dye particles in the cytoplasm. Superparamagnetic iron oxide-labeled The signal of mesenchymal stem cells in T2WI, especially in FFE (T2 * WI) sequence was significantly reduced. Under the concentration of 25mg / L Fe, the minimum cell number of MR imaging was 1 × 105. Under the different types of superparamagnetic iron oxide (P <0.01). There was no significant difference between T2WI and T2 * WI (P> 0.05) between USPIO and Feridex in T2WI and T2WI. Feridex-labeled mesenchymal stem cells showed statistically significant differences compared with T1WI at T2WI and T2 * WI (P <0.01). CONCLUSION: Superparamagnetic iron oxide can be easily labeled with mesenchymal stem cells and has no effect on the biological activity of mesenchymal stem cells at appropriate concentrations. MRT2WI and T2 * WI sequences can sensitively visualize magnetically labeled stem cells.