超顺磁性氧化铁标记对骨髓间充质干细胞多向分化诱导的影响

研究兔骨髓间充质干细胞(BMSCs)经超顺磁性氧化铁(SPIO)标记后,体外成骨、成脂及成软骨诱导能力的变化。体外贴壁培养和扩增兔BMSCs,采用SPIO(25μg/ml)联合硫酸鱼精蛋白转染剂标记兔BMSCs,分别采用适宜的成骨、成脂及成软骨诱导培养液对磁标记BMSCs进行体外定向诱导培养3周,诱导过程中观察细胞形态学变化。3周后对成骨定向诱导组进行钙结节茜素红染色和碱性磷酸酶(ALP)组化染色;对成脂肪定向诱导组行油红-O染色;对成软骨定向诱导组行番红O染色、II型胶原免疫组化染色检测观察胞外基质糖胺多糖、II型胶原的分泌和表达。利用Image-Pro Plus图像分析系统对免疫细胞化学染色进行光密度半定量分析。结果表明:超顺磁性氧化铁粒子标记BMSCs后普鲁士染色和电镜检查显示细胞胞浆内含致密铁颗粒;磁标记BMSCs在成骨、成脂及成软骨潜在多向分化诱导能力上与对照组未标记细胞相比无统计学差异。提示,SPIO联合硫酸鱼精蛋白转染剂能成功标记BMSCs,超顺磁性氧化铁标记对兔BMSCs体外多向分化诱导能力无明显影响,合理应用这种新型细胞标记技术将促进对组织工程种子细胞的研究。

Effects of superparamagnetic iron-oxide particles-labeling on the multi-diffentiation of rabbit marrow mesenchymal stem cell in vitro

The aim of this study was to label rabbit bone derived mesenchymal stem cells (BMSCs) with superparamagnetic iron oxide particles (SPIO) and to study the effects of magnetic labeling on the multi-differentiation of BMSCs. Rabbit BMSCs were isolated, purified, expanded, then coincubated with SPIO(25 microg/ml) complexed to protamine sulfate (Pro) transfection agents overnight. Prussian blue staining and transmission electron microscopy were performed to show intracellular iron. Cell differentiation was evaluated. Both labeled and unlabeled BMSCs were subjected to osteogenic, adipogenic and chondrogenic differentiation to assess their differentiation capacity for 21 d. Osteogenic cells were stained with alizarin red to reveal calcium deposition, adipogenic cells were stained with oil redO' respectively. Chondrogenic cells stained with Safranin-O, glycosamino glycans, and type II collagen production was assessed by standard immunohistochemistry. Cell with immunohistochemistry staining were detected by polarized light microscopy and analysed by Image-Pro Plus software. The results showed that intracytoplasmic nanoparticles were stained with Prussian blue and observed by transmission electron microscopy clearly except the unlabeled control. As compared with the nonlabeled cells, it showed no statistically significant difference on the differentiation of the labeled BMSCs. And the differentiation of the labeled cells were unaffected by the endosomal incorporation of SPIO. In summary, BMSCs can be labeled with SPIO without significant change in cell multi-differentiation capacity.