兔骨髓间充质干细胞的分离、培养与鉴定

目的:寻求家兔骨髓间充质干细胞(Bone Mesenchymal Stem cells,BMSCs)培养与分离简单易行的方法,为下一步骨髓间充质干细胞在呼吸系统疾病的应用打好基础。方法:取3个月龄家兔1只,经双侧髂前上棘抽取骨髓共5.0 ml,密度梯度离心法和贴壁筛选法相结合分离、纯化获得BMSCs,倒置差显微镜观察其形态学特性,流式细胞仪鉴定CD34、CD133表达。结果:接种细胞于24小时有少量细胞贴壁,72小时多数细胞可贴壁,贴壁细胞形态多为长梭型或多边形,原代细胞呈集落生长,12-16天可达90%融合,1%胰酶消化后传代培养,培养至第三代备用。流式细胞仪鉴定90%为骨髓间充质干细胞。结论:通过密度梯度离心法与贴壁筛选法可成功分离培养出骨髓间充质干细胞,此方法简单易行,成功率比较高。

Isolation,Culture and Identification of Bone Marrow-Derived Mesenchymal Stem Cells in Rabbits

Objective:To investigate a simple and feasible approach to isolating,culturing and identifying bone marrow-derived mesenchymal stem cells of rabbits.Methods:5.0 ml bone marrow was taken from bilateral anterior superior iliac spine of a rabbit aged 3 months and was isolated and purified by density gradient centrifugation and adherence screening method to obtain BMSCs.The morpho- logic features and cell number were observed by the inverted microscope.The expressions of CD44 and CD133 were determined by flow cytometry.Results:There were small amounts of cells adhereing 24 hours after culture and most of the cells adhereing 72 hours after cul- ture.Most of the adherent cells were long shuttle-shaped or polygon and the primary cells growed in clone.90% of the cells were fused on the 12th-16th days,after digested by 1% pancreatin,further cultured to 3rd generation to be prepared for use.About 90% pf the cells were verified to be BMSCs by flow cytometry.Conclusion:Bone marrow-derived mesenchymai stem cells of rabbits can be isolated and cultured successfully by the density gradient centrifugation and adherence screening method,which is simple and easy to be operated with higher successful rate.