成体狗骨髓间充质干细胞的分离和体外培养的初步研究

Research in separation and culture of mesenchymal stem cells(MSCs) from bone marrow of adult dogs.

目的探索成体狗骨髓间充质干细胞（MSCs）的分离、体外培养,为其将来应用提供实验依据。方法无菌条件下抽取狗肋骨骨髓,Ficoll分离液（密度1.077g/ml）梯度离心分离单个核细胞,在含10%新生牛血清的DMEM中,37℃、5%CO2、饱和湿度下常规培养,取贴壁细胞并进行传代,观察细胞的生长形态。结果取得较高纯度的成体狗骨髓MSCs,并保持细胞的活性;成体狗骨髓MSCs在体外培养中,为贴壁生长的单个核球形细胞,培养3-4d后开始大量增殖,为纺锤形和星形多突起的细胞贴壁生长,6-8d细胞达到70%-90%融合。传代细胞3-4d即可再次传代。结论采用Ficoll分离液进行梯度离心分离,可获较高纯度的MSCs,是实用、便捷和可行的方法;而且在体外培养的条件下能大量增殖,形成形态均一的细胞集落,可以成为进一步进行细胞扩增或其它实际应用的基础。

Objective To explore a practical method of the separation and the culture in vitro of mesenchymal stem cells （MSCs）from the bone marrow of the adult dogs,and to offer reference for its actual application. Methods The bone marrow was obtained from the fib of the adult dogs under the aseptic condition and was separated by gradient centrifugation in Ficoll（ density 1.077g/ ml） .The mononuclear cells were collected and cultured in DMEM with 10% new bovine serum. Adhering-wall cells were collected and continuously passage cultured. Results MSCs, separated from the bone marrow of the adult dogs, were collected in higher purity by of gradient centrifugation in Ficoll and still kept the cells activity. These cells,cultured in vitro,could grow in an adherent model on the wall and had a ball-like appearance at first. They were able to greatly proliferate in number after 3 - 4 days cultured and formed fusiform and multi-protuberance star shapes.After 6 - 8 days,they could reach 70% - 90% confluence. Subcultured cells could be cul- tured again after 3 - 4 days. Conclusion It is a simple and practical method to separate MSCs from the bone marrow of ad-t dogs by of gardient centrifugation in Ficoll. MSCs can greatly proliferate in number after a short period of the culture in vitro. Higher purity of MSCs,sepamted and cultured in vitro,will be very useful for its further research and application.