大鼠骨髓基质干细胞体外培养

A METHOD FOR LSOAATION AND CULTIVATION OF BONE MESENCHYMAL STEM CELLS BY BONE MARROE AND BYADHERING

目的:建立分离纯化、快速扩增大鼠骨髓基质干细胞(Bone mesenchymal stem cells,BMSCs)的方法。方法:用全骨髓接种贴壁法,培养分离纯化大鼠BMSCs,传代扩增、测定生长曲线和贴壁率,形态学观察、免疫细胞化学法鉴定细胞膜抗原。结果:全骨髓贴壁法能有效分离纯化大鼠BMSCs,细胞在10%胎牛血清BMSCs培养基中生长状态稳定,第2代细胞生长曲线呈S型、第2、4、6代贴壁率基本相同,贴壁速度无显著性差别,p>0.05。CD29、CD44和CD90免疫组化鉴定细胞均呈阳性。结论:全骨髓接种贴壁培养法能有效分离纯化大鼠BMSCs,在含10%胎牛血清的BMSCs培养液中,细胞稳定扩增,可能与细胞培养微环境良好有关。

Objective：To establish a method for isolation and cultivation of bone mesenchymal stem cells（BMSCs ） from rat bone marrow for offering an experimental foundation for actual application.Methods：Sacrifice the SD rats,separate and purify the BMSCs by whole bone marrow for adhering culture.After the in vitro subculture and expansion,observ atien of the living characteristic of the cells,draw mg the growth curve,were usde in order tomeasure the adhesive rate,study the morphology of the cells with phase contrast microscope and examine the cell surface antigen with immunocytochemistry technique.Results：The cells are able to stably grow in 10% BMSCs culture medium with 10%newborn bovine serum with the way of whole bone marrow and by adhering.The growth curve of second generation cells is S formation.The adhesive rate of 2,4 and 6 generation cells almost are same.Speed is not different specially,p 0.05.The cells are positive for the CD44,CD29 and CD90 immunostaining.Conclusion：The BMSCs could be effectively isolated and purified threugh density gradient centrifugation and adhering to the culture plastic and expanded satisfactorily in BMSCs culture medium with 10%newborn bovine serum.