摘要
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 。
To develop the methodology to purify and culture expand mesenchymal stem cells (MSC) from human bone marrow and investigate the optimal system for MSC differentiation into chondrocytes mononuclear cells were harvested by gradient centrifugation on Percoll at density of 1 073g/ml and seeded in low glucose DMEM containing 10% fetal calf serum. The purity of cells was evaluated by flow cytometric technique. MSC of 2 passages thereafter were absorbed into a biomaterial of gelatin sponge and induced to differentiate in to chondrocytes for one week under the influence of TGF β and other inductive agents. The feature of chondrocytes in engineered tissues was identified by toluidine blue staining at various time points. The results showed that human mesenchymal stem cells culture expanded were positive for CD166, CD29, and CD44, but negative for CD34, CD45, and HLA DR. Furthermore, when treated cells absorbed into the biomaterial were implanted subcutaneously into BALB c nude mice, they formed cartilage like tissues after 4 weeks.Our conclusions is that cultured marrow MSC have unique biological features and the capacity to differentiate into chondrocytes in vivo , so they are useful for cartilage engineering by serving as the seed cells.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2002年第12期1091-1093,F003,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家重点基础研究发展规划 (编号G1 9990 5430 2 )
国家自然科学基金(编号 30 0 70 857)资助课题
