人骨髓基质细胞培养及向成骨细胞的诱导分化

Human marrow stromal cells culture and differentiating into osteoblasts in vitro

目的研究人骨髓基质细胞体外培养及向成骨细胞诱导分化的实验方法。方法采用梯度离心法获得人骨髓基质细胞,细胞纯化后使用分化培养液将骨髓基质细胞向成骨细胞方向诱导分化。通过形态学观察、生化指标检测、细胞染色和矿化结节测定等方法,确定细胞的功能状态和分化程度。结果显微镜观察显示获得的人骨髓基质细胞生长状况良好,生化指标稳定;经分化培养液培养的细胞增殖速度明显减慢,生长状态平稳。细胞在分化培养过程中,上清液中碱性磷酸酶分泌量明显增加,细胞碱性磷酸酶染色明显浓染,随时间呈显著增强趋势;采用常规培养液培养的骨髓基质细胞,在汇合后不能形成明显的矿化结节。用分化培养液培养的人骨髓基质细胞,在14d时开始出现矿化结节,在21d时呈现密集的茜素红染色矿化结节。结论梯度离心法获得的人骨髓基质细胞生长情况良好,功能状态稳定;体外培养的人骨髓基质细胞在一定条件下可以向成骨细胞方向诱导分化,并具有良好的成骨细胞功能特征,可以满足进一步研究的需要。

Objective To observe and analyze the experimental condition of human marrow stromal cells （hMSC） culture and differentiating into human osteoblasts （hOB） in vitro. Methods The hMSC were obtained from bone marrow specimens by gradient centrifugation method. Then the cells were incubated with differentiation medium and differentiated along the hOB pathway. Morphology observation, biochemical detection and cells staining were performed during hMSC differentiation in vitro. Results The survival condition of the hMSC was good by morphological observation and biochemistry indexes were stable. The experimental data demonstrated that ALP secretion of differentiated hMSC increased significantly compared with undifferentiated cells. And the matrix secreted by differentiated hMSC began to mineralize after 14 days and increased significantly after 21 days. Conclusions Of the hMSC obtained, the survival condition was good, the biochemical indexes were stable. Under certain condition, hMSC can differentiate along the hOB pathway in vitro and the cellular function was excellent. They were fit for the next step study.