成人骨髓间充质干细胞培养条件优化及多向分化的能力

背景：成人骨髓中间充质干细胞数量少，要使其能够更好地应用于组织工程和细胞治疗，就必须建立成熟、简便、有效的分离培养扩增体系。 目的：建立成人骨髓间充质干细胞最佳培养方法，并观察诱导其向成脂肪、成骨及软骨细胞分化结果。 方法：分别采用密度梯度离心法和全骨髓培养法，分离培养成人骨髓间充质干细胞，通过光镜观察细胞形态，绘制生长曲线比较不同培养方法对骨髓间充质干细胞的影响；免疫荧光法鉴定表面抗原CD34、CD44和CD105的表达。不同诱导培养基诱导成人骨髓间充质干细胞，采用油红O、茜素红染色以及阿利新蓝染色检测成脂、成骨和成软骨诱导情况。 结果与结论：全骨髓培获得的骨髓间充质干细胞，其增殖能力和生长特性明显优于密度梯度离心法；细胞表面抗原CD44、CD105强阳性，CD34阴性；经过诱导培养，油红O、茜素红染色以及阿利新蓝染色均为阳性，证明全骨髓培养法获得细胞具有更强的生长增殖能力，并具有向脂肪，骨和软骨等组织多向分化的潜能。

Cultivation optimization and multi-directional differentiation potential of adult bone marrow mesenchymal stem cells

BACKGROUND: The number of adult bone marrow mesenchymal stem cells (BMSCs) is few. To apply BMSCs to tissue engineering and cell therapy, we should establish mature, simple and effective isolating culture amplification system. OBJECTIVE: To establish a method of isolation and cultivation in vitro of adult BMSCs, and to observe the results of BMSCs differentiating into the adipocytes, osteoblasts and chondrocytes. METHODS: Adult BMSCs were respectively isolated and cultivated by density gradient centrifugation and whole bone marrow culture. Cell morphology was observed under an optical microscope to draw growth curves. The effects of different culture methods on BMSCs were compared. CD34, CD44 and CD105 expressions were detected by immunofluorescence. Adult BMSCs were induced with different media. Differentiations of BMSCs into adipocytes, osteoblasts and chondrocytes were measured using Oil Red O staining, alizarin red staining and alcian blue staining. RESULTS AND CONCLUSION: By the method of whole bone marrow culture, the characteristics of proliferation and growth of BMSCs were superior to that by the method of density gradient centrifugation. Immunophenotypically, this cell population was found to be highly positive for CD44 and CD105, while negative for CD34. Following induced culture, Oil Red O staining, alizarin red staining and alcian blue staining were positive, which verified that BMSCs harvested by the method of whole bone marrow culture gained more capacity of growth and proliferation, and BMSCs could be induced to differentiate into osteoblasts, adipocytes and chondrocytes.