人骨髓基质细胞的分离、培养及成骨鉴定

ISOLATING AND CULTURING HUMAN BONE MARROW STROMAL CELLS IN VITRO AND IDENTIFYING THE OSTEOGENIC ACTIVITY

目的建立一种体外分离培养人骨髓基质细胞 (HumanBoneMarrowStromalCells ,hBMSCs)的方法。方法 :从髂棘 (髂前或髂后 )抽取骨髓液 5～ 8ml,经密度离心后的骨髓单核细胞 ,以 10 6/ml浓度接种培养 ,贴壁细胞接近长满后 ,进行传代培养 ,每传一代约 5～ 7d。培养期间进行碱性磷酸酶的检测和钙染色。结果 :原代培养和传代培养的细胞均为贴壁、形态不一的细胞。两种细胞在体外可向成骨方向转化。结论 :人骨髓基质细胞在体外长期培养后仍具有成骨能力 ,为骨髓中间充质干细胞 ,掌握其培养方法为其广泛应用奠定基础。

Objective:To estabish a method of isolating and culturing human bone marrow stromal cells(hMSCs) in vitro. Methods: A 5～8ml bone marrow was extracted from the iliac of human volunteers. The nucleated cells of marrow was enriched based on density centrifuge and cultured by 10 6/ml. The adherent cells approached confluence 12～14 days after plating and every passage was cultured for 5～7 days, during that time, the alkaline phosphatase (ALP) activity and Alizarin-Red staining were done. Results:The adherent cells during the first time and passaged several times in culture become different shapes in appearance, and they both can transform to osteoblast during long-time culture. Conclusions:The method for isolating and culturing hMSCs is foundmental and feasible, and these cells still possess the ability to differentiate into osteoblast after several passages.