目的建立一种新的高效体外骨髓间充质干细胞纯化培养方法,并对所获得细胞进行生物学行为研究。方法采用全骨髓培养获得原代骨髓间充质干细胞,经体外成脂、成骨诱导分化并检测细胞表面分子表达对所获得干细胞进行鉴定,传代后低密度接种(3×103/cm2)结合机械擦除对所培养细胞进行纯化,并对未纯化及纯化细胞分别进行集落形成单位实验、生长曲线及细胞周期分析来研究其生物学特性。结果全骨髓贴壁法可以成功培养出骨髓间充质干细胞,细胞呈现干细胞特性生长;可在体外成功地进行成脂、成骨分化;阴性表面标记分子CD34、CD45表达率均小于2%,阳性表面标记CD73、CD90表达率均高于95%。经纯化后的细胞具有更优的生物学行为,表现在:克隆形成率达到(28.80±5.00)%(P<0.01);培养12d后细胞数量增加(26.18±4.80)倍(P<0.05);处于S+G2/M期的细胞比例达到(45.98±1.60)%(P<0.01)。结论低密度接种结合机械擦除方法可以获得高纯度骨髓间充质干细胞,且该细胞具有更佳的生物学特性。 OBJECTIVE: To establish a new method for the purification and purification of bone marrow-derived mesenchymal stem cells in vitro and to study the biological behavior of the obtained cells. Methods Primary bone marrow mesenchymal stem cells (BMSCs) were obtained by whole bone marrow culture. The stem cells were identified by in vitro adipogenesis and osteogenic differentiation and cell surface molecular detection. After passage, the cells were seeded with low density (3 × 103 / cm2) The cultured cells were purified by erasing, and their biological characteristics were studied by colony forming unit experiment, growth curve and cell cycle analysis of unpurified and purified cells, respectively. Results Bone marrow-derived mesenchymal stem cells (BMSCs) were successfully cultured by whole bone marrow adherence method. The cells were characterized by stem cell growth. The adipogenic differentiation and osteogenic differentiation were successfully performed in vitro. The expression rates of negative surface marker molecules CD34 and CD45 were all less than 2% Surface markers CD73, CD90 expression rates were higher than 95%. The purified cells had better biological behavior. The results showed that the clone formation rate was (28.80 ± 5.00)% (P <0.01), the number of cells increased by 26.18 ± 4.80 (P <0.05) after cultured for 12 days. The proportion of cells in S + G2 / M phase reached (45.98 ± 1.60)% (P <0.01). Conclusion Low density vaccination combined with mechanical wipe method can obtain high purity BMSCs, and the cells have better biological characteristics.