骨髓基质干细胞的单克隆培养和分化的实验研究  

An experimental study on monoclonal culture and differentiation of marrow stromal stem cells

作　　者：郑东[1] 杨述华[1] 冯勇[1] 杨操[1] 李进[1] 梅荣成[1] 唐欣[1] 徐亮[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,湖北武汉430022

出　　处：《第四军医大学学报》2007年第15期1367-1371,共5页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30471753)

摘　　要：目的:通过对骨髓基质干细胞(MSCs)的单克隆化培养、鉴定和扩增,获得从一个单细胞克隆扩增出的大量均质的MSCs,并通过对这种MSCs诱导分化的实验研究,进一步确定这种单克隆来源的MSCs的分化和"横向分化能力".方法:无菌条件下获取大鼠的骨髓细胞,将获得的细胞经过充分打匀和滤过后制成单细胞悬液,然后以低密度,1×103个细胞数接种于培养板中,待细胞出现单细胞生长的克隆后,通过显微操作挑出相应的单细胞克隆.将挑出的单细胞克隆与相应的饲养细胞混合培养,快速进行扩增,扩增后的细胞进行细胞表面标志的鉴定,并进一步进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进行大量扩增,并保持细胞的均质性;而且在这种培养条件下,MSCs的增殖能力明显的高于常规培养条件;同时,获得的这种均质MSCs能成功的被诱导向软骨,神经样细胞分化.结论:通过骨髓细胞的单克隆培养,获得了均质和具有多向分化潜能的MSCs,这也为进一步更精确的研究MSCs的生物学特性提供了基础.AIM： To obtain a large number of homogeneous marrow stromal stem cells （MSCs） by monoclonal culture, identi- fication and proliferation of MSCs, and to investigate the differen- tiation and trans-differentiation potential of MSCs derived from the monoclone. METHODS： Single cell suspension was obtained after sufficient mixing and filtering of rat marrow cells and then seeded into culture plate at a low density （ 1-103 ）. Monoclonal cells were identified through microscope and were picked out with micromanipulative technique when these monoclones were obvious. Then these picked cells were co-cultured with mouse embryonic fibroblasts as feeder cells which were treated with myto- mycin for quick proliferation. The cells after proliferation were identified by means of cell surface markers. The monoclonal cells which contained the cell surface markers of MSCs were harvested and the potentials of proliferation and differentiation were explored further. RESULTS： MSCs derived from the monoclone prolifera- ted markedly without differentiation when they were co-cultured with feeder cells, and the proliferation was obviously quicker than that in the normal medium. At the same time such a co-culture maintained the homogeneous property of monoclonal MSCs. In ad- dition, the homogeneous MSCs had the potential to differentiate into chondrocytes and neuronal cells. CONCLUSION： MSCs derived from monoclone can be expanded massively when co-cul- tured with feeder cells, and have the potential to differentiate into chondrocytes and neuronal cells, which lays a foundation for fur-ther investigation of the biological properties of MSCs.

关 键 词：骨髓基质干细胞 克隆细胞 横向分化 

分 类 号：R318[医药卫生—生物医学工程]