论文部分内容阅读
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。
OBJECTIVE: To find a simple and convenient method for the culture and isolation of BMSCs in rabbits, and to lay the foundation for further application of BMSCs in respiratory diseases. METHODS: One rabbit of 3 months old was obtained. BMSCs were extracted from the anterior superior iliac spine by bilateral iliac spine. BMSCs were isolated by density gradient centrifugation and adherent screening. BMSCs were isolated and purified by inverted microscope. Morphological characteristics, Flow cytometry identification of CD34, CD133 expression. Results: Inoculated cells had a small number of adherent cells at 24 hours, most cells adhered at 72 hours, adherent cells were mostly spindle or polygons, primary cells grew colony, reached 90% confluence in 12-16 days, After 1% trypsin digestion and subculture, cultured to the third generation of spare. Flow cytometry identified 90% of bone marrow mesenchymal stem cells. Conclusion: BMSCs can be successfully isolated and cultured by density gradient centrifugation and adherence screening. The method is simple and easy to perform with high success rate.