Survival and growth of nano-bioprobe double-labeled rat bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2014.41.002

Abstract

Abstract:

BACKGROUND: Superparamagnetic iron oxide (SPIO) labeling can trace the migration of stem cells in vivo, and the fluorescent DiI dye is suitable for marking and tracing cells because of its less influence on cell viability, proliferation and differentiation.

OBJECTIVE: To observe the effect and safety of SPIO and fluorescent DiI dye to double label bone marrow mesenchymal stem cells．

METHODS: The bilateral lower limbs of rats were isolated sterilely. Bone marrow was obtained by rinsing using low-glucose DMEM. Bone marrow mesenchymal stem cells were isolated by the whole bone marrow adherence method and purified by differential attachment method. Purified cells were dual-labeled with SPIO particle and fluorescent DiI dye.

RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells could be separated at 8-10 days after primary culture and the subculturing cycle was 3-4 days. Rat bone marrow mesenchymal stem cells could be effectively labeled with SPIO-DiI and the labeling efficiency was almost 100%. Blue irons contained in intracytoplasmatic vesicles could be observed clearly with Prussian blue staining, and the fluorescence microscopy showed red fluorescence at cytoplasm. Survival and apoptosis percentages obtained by MTT analysis were similar among labeled and unlabeled bone marrow mesenchymal stem cells that were both about

95%．These findings indicate that the rat bone marrow mesenchymal stem cells could be efficiently labeled with SPIO-DiI to construct a nano-bioprobe, without significant changes in morphology, viability and proliferation.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: bone marrow, mesenchymal stem cells, cells, cultured, iron, carbocyanines

CLC Number:

R394.2

Cao Ai-hong,Yang Xin, Guo Zi-wei, Hu Wei. Survival and growth of nano-bioprobe double-labeled rat bone marrow mesenchymal stem cells [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(41): 6567-6572.

Figures/Tables 1

2.1 大鼠骨髓间充质干细胞培养纯化及形态学观察原代培养的细胞，48 h第1次换液见部分细胞贴壁，呈圆形或卵圆形，换液逐渐去除未贴壁细胞，全骨髓培养经过3-5 d、密度梯度离心法经过6-8 d的静止期，细胞逐渐伸出伪足，多数为成纤维样梭形或多边形细胞，大部分为单核，位于细胞中央，呈集落生长，细胞增殖迅速，集落相互靠近，慢慢融合，于培养9至10天(全骨髓培养法)、13-15 d(密度梯度离心法)渐渐铺满培养瓶。传代培养的细胞24 h完全贴壁，第1代的生长周期为五六天，随着传代次数增多，细胞形态越来越均匀，没有明显变化(图1)。传代后细胞四五天即可长满融合，以继续传代扩增。 2.2 流式细胞仪细胞表面标志测定流式细胞仪结果显示实验分离培养的骨髓间充质干细胞均一性好，达99%以上CD29+、CD90+、CD45-，说明传代贴壁生长的梭形细胞确为骨髓间充质干细胞。 2.3 骨髓间充质干细胞多能横向分化潜能鉴定成脂肪诱导：诱导3 d后，细胞由成纤维细胞样逐渐收缩变短，成为立方形或多角形；连续培养7 d，镜下可见细胞内有微小脂滴出现，随着时间的延长，脂滴逐渐增大融合，培养2周时，可见融合成团的脂滴充满整个细胞，油红O染色，可见细胞内大量红色脂滴。成骨诱导：培养1周后，细胞形态明显改变，由成纤维细胞样变为多角形，类似神经元细胞，细胞周边出现长丝状突出，向周围延伸。培养2周以上，细胞基质矿化物逐渐出现，培养4周以上，可见明显钙化结节，von Kossa染色可见钙盐沉积区域呈黑色。 2.4 Fe2O3-PLL磁性纳米粒子的表征分析将合成的产物经透射电镜观察，nano-Fe2O3-PLL呈球形，分布均匀。直径分布为10-20 nm，平均直径为16 nm，具有立方尖晶石的晶体结构。饱和磁化强度为60 emu/g。 2.5 SPIO-DiI双标记后细胞内铁及荧光染料检测标记后的活细胞在倒置相差显微镜下已能见到胞浆内的棕黄色颗粒(图2A)，细胞爬片普鲁蓝染色后，大鼠骨髓间充质干细胞的几乎每个标记细胞浆内均可看到蓝色颗粒，标记率近100%(图2B)，而未标记细胞内无蓝色颗粒。荧光显微镜观察，可见到红色环状荧光，红色颗粒为细胞膜及细胞浆，中间未染色的为不着色的细胞核，几乎每个标记细胞均可看到红色荧光，标记率近100%(图2C)。 2.6 双标对骨髓间充质干细胞活力及增殖的影响锥虫蓝染色计数显示移植前标记和未标记活细胞数目均在95%左右(表1)，说明在含SPIO-DiI的培养基中骨髓间充质干细胞可继续增殖，细胞形态无明显变化。"

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