小型猪骨膜干细胞的分离与鉴定

Isolation and Identification of Periosteal Derived Stem Cells in Miniature Pigs

目的:研究哺乳动物的骨膜中是否存在间充质干细胞。方法:取1月半龄的贵州小香猪髂骨近端内侧的骨膜,经I型和II型胶原酶消化分离骨膜细胞,在标准条件下体外细胞培养约3周,加入抗CD90,CD44,CD105,CD14,CD45的单克隆抗体孵育后,经流式细胞学分析;再通过干细胞分化试剂盒诱导成骨、成软骨和成脂分化,鉴定其干细胞特性。结果:从猪骨膜分离消化的细胞,经体外培养3周后,细胞呈梭形或不规则三角形生长,胞质向外伸出长短不同的突起;流式细胞学分析显示从骨膜分离的细胞能表达表面抗原CD44、CD90和CD105,而不表达CD14和CD45;经诱导该细胞能向成骨、成软骨和成脂分化。结论:本研究成功分离并鉴定了小型猪骨膜来源的干细胞(Periosteal derived stem cells,PDSCs),为软骨缺损修复的组织工程技术提供了种子细胞来源。

Objective To test whether there are mesenchymal stem cells in the periosteum of pigs.Methods Periosteal cells were isolated after the periosteum of the proximal medial side of the sacrum of pigs was taken and digested with type I and type II collagenase. Then these cells were cultured in a standard condition for about 3 weeks. After monoclonal antibodies against CD90, CD44, CD105,CD14 and CD45 were added for incubating,they were evaluated using the flow cytometry. Later the stem cell differentiation kits(toward osteogenic, chondrogenic, and adipogenic differentiation) were used to identify their characteristics. Results After culturing in vitro for three weeks,spindle or irregular triangle cells were visible,and the cytoplasm protruded outwards with processes of varying lengths.Flow cytometry showed that cells isolated from periosteum could express surface antigens of CD44,CD90,and CD105,but not CD14 and CD45. Moreover,they could be induced towards osteogenic,chondrogenic and adipogenic differentiation. Conclusion In this study, we have successfully isolated and identified periosteal derived stem cells of miniature pigs,which can be sources of seed cells to repair the cartilage defect using tissue engineering techniques.