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为建立分离纯化、大量扩增人骨髓间充质MSC ,改进其冰冻保存的方法 ,并初步探索其体内向软骨细胞分化的最佳方法 ,以Percoll(1 0 73g/ml)密度梯度离心法分离人骨髓中单个核细胞 ,用含经筛选的 10 %的胎牛血清的低糖DMEM体外培养扩增骨髓干细胞(MSC) ,FCM鉴定细胞纯度 ,传代 2次以后的细胞吸附于明胶海绵内 ,以TGF β为主要刺激因子体外诱导 1周 ,植入裸鼠皮下。在不同的时间取出组织块 ,甲苯胺蓝染色显示细胞外基质。结果发现 ,体外培养扩增的人间充质MSC表达CD166、CD2 9、CD44等表面抗原 ,不表达CD3 4、CD45、HLA DR等抗原 ;MSC可以不经消化 ,直接在原培养瓶内冻存在 -70℃低温冰箱 ,3个月后复苏的细胞生物学特性没有明显改变 ;体外诱导的细胞植入裸鼠皮下 4周后即出现软骨细胞特有的结构。说明骨髓MSC具有独特的生物学特征 ,体外培养的MSC具有体内成软骨能力 ,应用MSC构建软骨组织具有一定的可行性
In order to establish a method for isolation and purification of human bone marrow mesenchymal stem cells (MSCs), and to improve their cryopreservation, the best way to differentiate into chondrocytes in vivo was established. Percoll (1073g / ml) density gradient centrifugation Human bone marrow mononuclear cells, bone marrow stem cells (MSC) were amplified by in vitro culture with low glucose DMEM containing 10% fetal bovine serum. FCM was used to identify the purity of the cells. After passage 2, the cells were absorbed in gelatin sponge. β as a major stimulus for 1 week in vitro, implanted subcutaneously in nude mice. Tissue pieces were removed at different times and toluidine blue staining showed the extracellular matrix. The results showed that cultured MSCs cultured in vitro expressed CD166, CD2 9, CD44 and other surface antigens, but did not express CD3 4, CD45, HLA DR and other antigens; MSC could be stored in the original culture flask without digestion -70 ℃ low temperature refrigerator, three months after the recovery of the cell biological characteristics did not change significantly; in vitro induced nude mice subcutaneously 4 weeks after the cartilage cells that appear unique to the structure. Explain the unique biological characteristics of bone marrow MSCs, MSC cultured in vitro has the capacity of cartilage in vivo, the application of MSC construction of cartilage tissue has a certain feasibility