Wistar大鼠骨髓间充质干细胞培养、鉴定及标记.docVIP

Wistar大鼠骨髓间充质干细胞培养、鉴定及标记 　作者：赵科研 柳克祥 侯明晓 孙江滨 吴慧颖 李博 【摘要】 　　目的 建立Wistar大鼠骨髓间充质干细胞（MSCs）分离、培养的方法，并进行鉴定、标记。方法 采用全骨髓贴壁培养法分离培养大鼠的MSCs。采用相差显微镜观察MSCs的形态学特征；流式细胞仪检测第3代细胞表面标志抗原CD29、CD44、CD34和CD45的表达率；电镜检查第3代MSCs超微结构。DAPI标记MSCs为下一步进行体内细胞移植示踪。结果 原代培养的MSCs于6～8 h后贴壁，6 d左右形成集落，14 d 左右可达到80%～90%融合。第3代细胞表面标记物CD29，CD44，CD34和CD45的阳性率分别为98.6 %，78.2%，0.0%，0.3%。超微结构清晰，可见细胞器，如线粒体、粗面内质网和高尔基复合体，细胞核呈圆形或不规则，可见较多微绒毛。DAPI可良好标记MSCs细胞核，呈蓝色荧光。结论 采用全骨髓贴壁培养的方法能够成功分离和培养大鼠的MSCs，在第3代可获得高纯度MSCs。DAPI可以作为一种示踪剂标记MSCs。 【关键词】 细胞培养；间充质干细胞；种子细胞；示踪剂 【Abstract】 Objective To establish a method for isolation and culture of bone marrow mesenchymal stem cells(MSCs) from Wistar rats in vitro,and to identify characteristic of the cells and to label them after culture expansion.Methods MSCs were isolated and cultivated from the bone marrow of Wistar rats by adherent method.The morphology of MSCs was observed under phase contrast microscope.The expression of CD29 , CD44 , CD34 and CD45 of the third generation cells were analyzed by flow cytometry. Electron microscopic features were also observed and MSCs were labeled by DAPI. Results After 6 to 8 h primary culture , MSCs adhered to plastic surface of the culture dish. 　About 6 d later , the cells proliferated in colonies.Primary MSCs reached 80%～90%of confluence in 14 d approximately.The positive rates of CD29 , CD44 , CD34 , and CD45 in MSCs at third generation were 98.6% ,78.2% ,0.0% , and 0.3% respectively. Under the electron microscope MSCs had plentiful cytoplasm with mitochondria rough endoplasmic reticula and Golgi complexes.Their nuclei were round or irregular and there were plenty of microvilli on the surface. All of the MSCs after labeling by DAPI showed blue fluorescence by fluoroscope.Conclusions Mesenchymal stem cells can be successfully isolated and cultivated by adherent method.And higher purity MSCs can be picked up after culture expansion at P3. DAPI can be an effective tracer agent to label MSCs. 【Key words】 Cell culture;Mesenchymal stem cells;See