摘要
目的:骨髓基质细胞由处于不同分化阶段的多种细胞组成,一般认为它包括成骨细胞、成纤维细胞、脂肪细胞、网状细胞及其干细胞,为一异质细胞群,其中的干细胞具有多向分化能力.直接应用MSC修复关节软骨,新生透明样软骨容易发生退变,其异质性是重要原因之一.体外培养条件下诱导MSC分化为较为单一的软骨细胞将有助于提高新生软骨的质量及促进其表型维持.Ephrat采用高密度培养技术诱导MSC分化为软骨细胞,但是一部分细胞分化为成骨细胞,体外形成钙化结节.所用培养液含10%FRS、VitC50ng/ml\Dex0.4μg/ml\bFGF25mg/ml.本研究调整培养液,使MSC分化为软骨细胞.方法:抽取新西兰大耳白兔骨髓于DMEM中培养扩增,DMEMd培养液中含10%FRS,VitC(50ng/ml).青霉索(100u/ml),链霉素(100u/ml).之后高密度(1×10~6cells/cm^2)和低密度(1×10~4celts/cm^2)传代培养,观察细胞形态变化,甲苯胺兰、ALP染色,I、Ⅲ型胶原和S-100免疫组化染色以判断MSC的表型转化.收集细胞植入BALB、C-nu/nu品系裸鼠皮下观察骨、软骨形成情况.结果:低密度传代后MSC贴壁生长,形成CFU-F.呈成纤维细胞样,甲苯胺兰染色阴性,Ⅲ型胶原阳性,植入裸鼠皮下3w形成骨软骨组织结节.高密度传代后90-95%IVEC甲苯胺兰染色阳性,S-100阳性,I、Ⅲ型胶原阴性.具有软骨细胞的功能表型;植人裸鼠皮下形成软骨组织性结节,没有骨组织形成.结论:在本实验条件下,用不含Iex、bFGF的DMEM高密度传代培养可诱导骨髓基质细胞分化形成软骨细胞,未见钙化结节的形成.ALP染色阴性,未测出有骨钙素合成,获得MSC源性软骨细胞.
Aim: The bone marrow stromal cell (MSC) is a differentiationally heterogenous cell group, including reticular cells, adipocytes, fibroblasts, osteogenic cells and stem cells, among which the latter is pluripotent. The heterogenity is one of the important reasons causing degradation of the new haline-like cartilage formed in MSC-based repair of articular cartilage. MSC may be induced to differentiate into chondrocyte in vitro. This will improve the quality of new cartialge and maitain its phenotype. Methods: The bone marrow was aspirated from the proximal tibia of New Zealand White rabbits, and cultured for propagation in DMEM, containing 10% FBS, VitC 50ng/ml, penicillin 100~u/ml, and streptomycin 100~u/ml. When confluence was achieved after about 2 weeks, the cells were subcultured at two initial cell density: 1 × 10~4 cells/cm^2(low cell density), 1 × 10~6 cells/cm^2 (high cell density) under the same environmental conditions. The presence of chondrocytes in the subcultures was assessed by means of histological staining with toluidine blue, AIP and by means of immunological straining for type I, III collagen, S-100 protein and by means of subcutaneous chondrogenesis in BAIB/c-nu/nu mice. Results: CFU-F was formed at low cell density subculture, being negative for toluidine blue and type I collagen, positive for type IIIcollagen, forming node in mice containing bone and cartilage 3 weeks atter transplantation; a fibroblast monolayer was formed at high initial cell density, 90-95% cells were positive for toluidine blue and S-100 stairing negative for type I and III collagen, forming cartilaginous node without esseous tissue. Conclusion: Formation of MSC derived chondrocytes is achieved at high initial cell density when cultured under the present environment.
出处
《福州总医院学报》
1999年第1期15-18,共4页
Journal of Fuzhou General Hospital
