| 李俊义,徐华林,万京明,姜 彦,李 娜.猫骨髓间充质干细胞的体外分离培养、纯化、鉴定[J].,2018,(1):48-52 |
| 猫骨髓间充质干细胞的体外分离培养、纯化、鉴定 |
| Isolation, Cultivation and Identification of Cat Bone Marrow Mesenchymal Stem Cells in Vitro |
| 投稿时间：2017-06-11 修订日期：2017-07-06 |
| DOI：10.13241/j.cnki.pmb.2018.01.010 |
| 中文关键词: 家猫 骨髓间充质干细胞 分离培养 鉴定 |
| 英文关键词: Cat Mesenchymal stem cells Isolation and cultivation Identification |
| 基金项目:国家自然科学基金项目(81170895)；山东省自然科学基金项目(ZR2009CM053) |
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| 中文摘要: |
| 摘要 目的：分离、培养、纯化家猫的骨髓间充质干细胞，并对获得细胞的表面标志物进行鉴定，为进一步利用骨髓间充质干细胞的细胞移植实验奠定基础。方法：采用全骨髓贴壁法体外分离、培养、纯化家猫骨髓间充质干细胞，通过多次更换培养液获得较纯化的骨髓间充质干细胞，倒置相差显微镜下对细胞形态进行观察；根据第1、3、5、7、9代细胞的镜下增殖情况绘制出生长曲线；通过流式细胞仪检测细胞表面标志抗原CD34、CD44和CD90的表达率。结果：在倒置相差显微镜下观察，分离培养的骨髓间充质干细胞贴壁呈梭形或纺锤形；原代细胞生长丛集成片，5～7 d 达到融合，进行传代；培养到第三代以后，细胞出现相对均匀的梭形扁平外观，迅速增殖的细胞呈涡流样排列；第3、5代骨髓间充质干细胞增殖能力强于第7、9代；采用流式细胞仪分析结果显示细胞的CD34、CD44和CD90阳性率分别为17.5%、97.9%和91%，这与骨髓间充质干细胞表面抗原的表达一致。结论：分离培养的细胞具有骨髓间充质干细胞特性，成分相对单一，第3、5代细胞纯度高，增殖能力强，适用于进一步的实验研究。 |
| 英文摘要: |
| ABSTRACT Objective: To establish a method to isolate and culture cat bone marrow mesenchymal stem cells in vitro and analyze their phenotypical properties after culture expansion as well as preliminary identification, so as to establish foundation for the further study of bone marrow mesenchymal stem cells transplantation. Methods: Bone marrow mesenchymal stem cells from cats were isolated, cultured and purified by the whole bone marrow adherence method. The bone marrow mesenchymal stem cells were purified by discard- ing suspended cells through exchanging medium. The morphology of cultured bone marrow mesenchymal stem cells was observed under inverted phase contrast microscope. The growth curve of cell proliferation was obtained based on the observation of the proliferation sta- tus of 1st, 3rd, 5th, 7th, and 9th generation. The expressions of CD34 and CD44 and CD90 of cells were analyzed by using flow cytome- try in order to identify bone marrow mesenchymal stem cells. Results: Bone marrow mesenchymal stem cells had spindle shape under in- verted phase contrast microscope. The primary cells were confluent in single layer after being plated for 5-7 days, and then the cells were passaged. After extending to the third passage, cells appeared relatively uniform appearance with fusiform or flattened, which were rapid proliferated as arranged with swirl observed under inverted phase contrast microscope. Bone marrow mesenchymal stem cells of 3rd, 5th generation proliferated more rapidly than those of 7th and 9th generation. Flow cytometric analysis demonstrated the positive rate of CD34, CD44, CD90 were 17.5%, 97.9%, 91%, respectively, which was consistent with the surface antigen of bone marrow mesenchymal stem cells. Conclusion: Purified bone marrow mesenchymal stem cells can be obtained via this protocol. Cells of 3rd, 5th generation with high proliferation are fit to the further experiment. |
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