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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/mlDex0.4μg/mlbFGF25mg/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.
OBJECTIVE: Bone marrow stromal cells consist of a variety of cells at different stages of differentiation. It is generally considered to include osteoblasts, fibroblasts, adipocytes, reticulocytes and stem cells thereof as a heterogeneous population of cells in which stem cells have many To the differentiation ability of the direct application of MSC repair articular cartilage, new transparent hyaline cartilage prone to degeneration, the heterogeneity is one of the important reasons.In vitro culture conditions, the differentiation of MSC into a single chondrocyte will help improve the newborn cartilage Ephrat induces MSCs to differentiate into chondrocytes using high-density culture, but some cells differentiate into osteoblasts and form calcified nodules in vitro using 10% FRS, VitC 50 ng / ml Dex 0.4μg / ml bfGF25mg / ml.In this study, the culture medium was adjusted to differentiate into chondrocytes.Methods: The bone marrow of New Zealand white rabbits was cultured and expanded in DMEM, DMEMd medium containing 10% FRS, VitC (50ng / ml), penicillin (100u / ml) and streptomycin (100u / ml), followed by high density (1 × 10 ~ 6cells / cm ~ 2) subculture, observe the morphological changes of cells, toluidine blue, ALP staining, I, Collagen and S-100 immunohistochemical staining to determine the phenotypic transformation of MSCs. BALB / C nude nude mice were collected and subcutaneously injected with BALB / C nude mice to observe the formation of bone and cartilage.Results: , Formed CFU-F fibroblast-like, negative for toluidine blue stain positive for type III collagen, and subcutaneously implanted into nude mice for 3 weeks to form osteochondral nodules.After 90-95% IVEC positive staining for toluidine blue with high density , Positive for S-100, negative for type I and type III collagen, functional phenotype of chondrocytes, formation of cartilage tissue nodules subcutaneously in nude mice without formation of bone tissue.Conclusion: Under the experimental conditions, , BFGF DMEM high-density subculture can induce the differentiation of bone marrow stromal cells into chondrocytes, no formation of calcified nodules.ALP staining was negative, no osteocalcin synthesis was detected, and MSC-derived chondrocytes were obtained.