骨髓基质干细胞的单克隆化培养和多向分化的实验研究

An Experimental Study on Monoclonal Culture and Differentiation of Marrow Stromal Stem Cells

目的通过对骨髓基质干细胞(marrow stromal cells,MSCs)的单克隆化培养、鉴定和扩增,获得从一个单细胞扩增出的大量均质MSCs,并研究其多向分化能力。方法无菌条件下获取大鼠骨髓细胞,在培养的过程中挑出相应的单细胞克隆。将挑出的单细胞克隆与饲养细胞混合培养,快速进行扩增和细胞表面标志的鉴定,并进一步行增殖和细胞分化实验。结果在含有特定饲养层细胞的培养条件下,单克隆来源的MSCs能在抑制分化的状态下大量扩增,并保持细胞的均质性;而且在这种培养条件下,MSCs的增殖能力明显增强,并能成功被诱导向软骨、神经样细胞分化。结论通过骨髓细胞的单克隆培养,获得了均质和多向分化潜能的MSCs,为进一步更精确的研究MSCs的生物学特性提供了基础。

Objective To obtain a large number of homogeneous marrow stromal stem cells （MSCs） by monoclnal culture, identification and proliferation of marrow stromal stem cells. Meanwhile, the differentiation and trans--differentiation potential of MSCs derived from the monoclone were investigated. Methods Single cell suspension was obtained after plentiful mixing and tilting of rat marrow cells and then Seeded intd culture flask at a low density. Monoclonal cells were identified through microscope and were picked out when these monoclones were obvious by micromanipulative technique. Then these picked cells were co-cultured with mouse embryonic fibroblasts as feeder cell which were treated with mytomycm for quickly proliferation. The cells after proliferation were identified by means of cell surface markers. The monoclonal cells which contain the cell surface markers of MSCs were harvested and the potential of proliferation and differentiation were explored further. Results MSCs derived from the monoelone could be able to prohferate markedly without differentiation when they were co-cultured with feeder cells, and the prohferation obviously quicker than that compared with the normal medium. At the same time co-culture maintain the homogeneous property of monoclonal MSCs. In addition, the homogeneous MSCs have the potential to differentiate into chondrocytes and neuronal cells. Conclusion MSCs derived from monoclone can be expanded massively when co-cultured with feeder cells, and have the potential to differentiate into chondrocytes and neuronal cells that lays a foundation for further investigation of the biological properties of MSCs.