大鼠骨髓间充质干细胞的体外分离培养与鉴定  

Isolation and identification of mesenchymal stem cells in vitro

作　　者：何继业[1] 王栋梁[1] 董海[1] 彭建平[1] 陈晓东[1] 

机构地区：[1]上海交通大学医学院附属新华医院骨科,200092

出　　处：《中华手外科杂志》2008年第3期174-177,共4页Chinese Journal of Hand Surgery

基　　金：国家自然科学基金资助项目（30471748）

摘　　要：目的体外观察大鼠骨髓间充质干细胞的基本生物学特性，建立rMSCs的分离培养方法，为诱导分化成肌研究打下基础。方法采用Percoll密度梯度离心法分离纯化大鼠MSCs。观察细胞的形态和生长特性，倍增时间，生长曲线，用流式细胞仪分析rMSCs的表面抗原。结果体外培养的rMSCs很快贴壁，4-8d即贴壁80％，细胞增殖迅速，7d左右即可达到80％融合。流式细胞仪分析CD90、CD13、CD44表达强阳性，CD14、CD34、CD45和HLA-DR表达阴性，符合MSCs的表面抗原特征。结论经Percoll密度梯度离心法获得的rMSCs细胞株较纯，可作为组织工程中的种子细胞来源，但大鼠MSCs在体外培养过程中易于衰老。Objective To observe the biological characteristics of rat mesenchymal stem ceils （rMSCs）, establish a protocol for rMSCs isolation and culture, and provide basis for further myoblast differentiation research. Methods Rat MSCs were isolated and purified using Percoil density gradient centrifugation. Morphology and growth features of the cells were observed to obtain the proliferation time and growth curve. Surface antigens were identified by flow cytometry. Results The harvested rMSCs attached to flask wall easily, 80% of the cells were attached between 4 to 8 days of culture. The ceils showed fast proliferation and reached 80% confluency at day 7. Flow cytometry showed positive CD90, CD13 and CD44 and negative CD14, CD34, CD45 and HLA-DR, the antigen phenotypes conforming to typical MSCs. Growth curve showed rMSCs at primary culture, P1 and P2 proliferate rapidly; it slowed down at P3, and after P4, the proliferation was remarkably decreased. Conclusion rMSCs harvested by Percoil density gradient centrifugation have high purity and are ideal seed cells for tissue engineering. The cells however age in the process of in vitro culture.

关 键 词：大鼠 肌细胞 细胞 培养的 鉴定 间充质干细胞 

分 类 号：R686[医药卫生—骨科学]