Effect of CM-Dil labeling on biological characteristics of rat bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2016.45.006

Abstract

Abstract:

BACKGROUND: Existing evidence has shown that CM-Dil-labeled mesenchymal stem cells have delivered better results in experiments in vivo and in vitro.
OBJECTIVE: To investigate the effect of in vitro CM-Dil labeling on biological characteristics and directed migration of rat bone marrow mesenchymal stem cells.
METHODS: Mesenchymal stem cells were isolated and cultured from the bone marrow of Sprague-Dawley rats. The specific surface antigens, CD29, CD44, CD11b, CD45, were detected by flow cytometry. The rat bone marrow mesenchymal stem cells were labeled with CM-Dil, and then were cultured and passed continually. Thereafter the morphology and proliferation ability of labeled cells were assessed. The strength and duration of fluorescence after CM-Dil labeling were detected. Transwell chambers were used to investigate the migration ability of labeled cells towards the homogenate of the gastrocnemius muscles of mdx mice.
RESULTS AND CONCLUSION: The cultured cells were markedly positive for CD29 and CD44, but negative for CD11b and CD45, which were in accordance with the features of bone marrow mesenchymal stem cells. CM-Dil-labeled cells were not different from unlabeled cells in the aspect of morphology and proliferation. Fluorescence was still visible after passage; however, a reduction in fluorescent intensity was observed under an inverted fluorescent contrast phase microscope after labeling. The labeled cells showed no different migration ability from the unlabeled cells (P > 0.05). In conclusion, the procedure of CM-Dil labeling is safe, simple and effective. CM-Dil could be a good choice for labeling and tracing rat bone marrow mesenchymal stem cells.

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

Key words: Stem Cells, Bone Marrow, Cell Proliferation, Tissue Engineering

CLC Number:

R394.2

Zhang Ya-ni, Yu Mei-juan, Feng Shan-wei, Wang Shu-hui, Li Mei-shan, Xiong Fu, Zhang Cheng. Effect of CM-Dil labeling on biological characteristics of rat bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(45): 6726-6732.

Figures/Tables 1

2.1 大鼠骨髓间充质干细胞的形态与生长特点以差速贴壁法原代培养的大鼠骨髓细胞，在刚分离接种时镜下可见大量密集悬浮细胞，接种后三四小时，细胞开始少量贴壁，在瓶底散在分布；8-12 h细胞约60%贴壁；24 h后约80%细胞贴壁，多呈扁平形、梭形或多角形，其中有大鼠骨髓间充质干细胞也有杂质细胞，悬浮细胞多呈圆形，大部分为造血干细胞和血细胞，生存寿命较短，两三次换液后可基本清除。3 d后细胞增殖明显，形态趋于纺锤状，5-7 d可见细胞集落，10-14 d细胞融合，多数呈均一典型的“纺锤状”成纤维样细胞形态；细胞间排列呈“旋涡状”、“辐射状”或“栅栏状”。刚传代的细胞呈圆形，6-8 h内又可贴壁、伸展重新成为梭形细胞；24 h后开始增殖，细胞集落增多；7-10 d后可传代，每传一代细胞数量可增加约2.5倍。P3代以上的大鼠骨髓间充质干细胞趋于纯化，细胞呈均质状，细胞增殖融合密集排列呈束，有一定方向性，呈集落形式生长。一般应用P5代作后续实验(图1)。 2.2 大鼠骨髓间充质干细胞表面标记物的检测结果流式细胞检测显示，体外培养传代贴壁生长的P5代大鼠骨髓间充质干细胞均一性好，纯度达95%以上。大鼠骨髓间充质干细胞表面CD11b和CD45检测阴性，而CD29和CD44表达阳性。 2.3 CM-Dil标记大鼠骨髓间充质干细胞后的形态观察及荧光强度追踪在相差显微镜下观察5 μmol/L浓度标记P5代细胞后其形态与未标记细胞相比未见明显差异。荧光显微镜下以绿色荧光激发标记细胞后，可见胞浆均匀发出强烈的红色荧光，细胞核无荧光发出，经流式细胞仪检测标记率>98%。体外传代至P6，P7代仍可检测到红色荧光，但随着体外传代荧光强度出现一定程度减弱(图2)。 2.4 标记后大鼠骨髓间充质干细胞的细胞生长曲线经CM-Dil标记与未经标记的P3代细胞生长曲线大致相似，在培养1 d时细胞量稍有减少，为细胞适应期，3 d后细胞数迅速增长，至第8天进入平台期，细胞增殖减慢(图3)。 2.5 体外Transwell细胞迁移能力 CM-Dil标记后的P5代大鼠骨髓间充质干细胞[(407±20)个/视野]向mdx鼠腓肠肌匀浆液的迁移能力与未标记细胞[(417±27)个/视野]相比差异无显著性意义(P > 0.05)，标记细胞向mdx鼠腓肠肌匀浆液的迁移能力[(407±20)个/视野]显著高于其向空白液迁移能力[(378±16)个/视野，P < 0.01]。"

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