兔骨髓间充质干细胞的筛选与体外培养

目的 :探讨兔骨髓间充质干细胞 (mesenchymalstemcells ,MSCs)体外分离筛选及扩增的条件 ,同时观察骨髓细胞体外培养的生物学特性。方法 :无菌条件下采集兔骨髓 ,肝素抗凝 ,经淋巴细胞分层液 (相对密度为 1 .0 77)密度梯度离心分离骨髓单个核细胞 ,进而贴壁培养 ,获得MSCs。采用常用细胞培养技术和细胞培养的研究方法 ,观察不同贴壁时间、不同种植密度、有无包被成分及不同蛋白包被培养板对MSCs生长增殖的影响。结果 :预先蛋白包被培养板有利于MSCs贴壁。在细胞生长和增殖方面 ,纤粘连蛋白优于明胶和Ⅰ型胶原 ,以贴壁 48～ 72h ,种植密度 (3～ 9)× 1 0 4 /ml为最适条件 ,有利于梭形细胞形态和快速增殖能力的维持 ,保持成体干细胞的多向分化性。结论 :建立了MSCs体外分离和培养的最适条件 ,为进一步研究MSCs的诱导分化和应用提供了保证

Isolation and in vitro expansion of rabbit bone marrow mesenchymal stem cells

Objective This study is designed to explore the isolation,cultivation of bone marrow mesenchymal stem cells(MSCs) in vitro. Methods Rabbit bone marrow was aspirated from the iliac crest sterilely with heparin. Bone marrow mononuclear cells were isolated by gradient density centrifugation with lymphocyte separation medium.Mesenchymal stem cells(MSCs) were then obtained and cultured in completely chemically defined medium.Common cell culture technique and normal methods were used to study the effects on the growth,proliferation,morphology of MSCs in different pre-adsorbed protein coated plastic plate,adherent time and cell density. Results The optimal condition in vitro for growth was pre-adsorbed protein coated on the plastic bottom ,48～72 hours adherent time,(3～9) 10 4 /ml cell density.Spindle cells had a potential ability of proliferation and pluripotent differentiation. Conclusion The optimal condition for isolation and culture of bone marrow MSCs were successfully established.It is a base for further investigation and use of MSCs.