绵羊骨髓间充质干细胞分离培养与鉴定

Isolation,culture and identification of sheep bone marrow mesenchymal stem cells

旨在分离绵羊骨髓间充质干细胞(sheep bone marrow mesenchymal stem cells,SBMSC),建立绵羊骨髓间充质干细胞系,为相关研究提供种子细胞。取新生健康绵羊肋骨为试验材料,利用贴壁法分离获得SBMSC;取生长良好的P3代细胞通过免疫荧光,进行CD73、CD90、CD105、CD106表型鉴定;取生长良好的P3代细胞分别进行成软骨、成脂、成骨方向的诱导,对诱导结果分别进行阿利新蓝、油红O和茜素红染色,鉴定分化结果。SBMSC呈成纤维样贴壁生长;体外培养P3代细胞表面标记物CD73、CD90、CD105和CD106完全符合标准;P3代细胞经软骨、脂肪、骨方向诱导3周后,经阿利新蓝染色,细胞呈亮蓝色;经油红O染色,细胞质内出现橙红色脂滴;茜素红染色表明聚集的细胞团中央能形成钙化结节。说明SBMSC经体外诱导培养后可向软骨细胞、脂肪细胞和成骨细胞分化,并具有明显的成软骨、成脂和成骨表型,表明SBMSC具有多向分化潜能。通过贴壁培养法建立了SBMSC完整的体外培养体系,为绵羊育种等方面提供种子细胞。

This study aims to isolate sheep bone marrow mesenchymal stem cells(SBMSC)and establish sheep bone marrow mesenchymal stem cell lines to provide seed cells for related studies.The ribs of newborn healthy sheep were used as experimental materials,and SBMSC was obtained by adherent method.CD73,CD90,CD105 and CD106 phenotypes were identified by immunofluorescence in P3 cells with good growth.The P3 cells with good growth were induced in chondrogenic,adipogenic and osteogenic directions,respectively,and the results were stained with Alcian blue,Oil Red O and alizarin red,respectively,to identify the differentiation results.SBMSC grew as fibrous adherent.The identification of CD73,CD90,CD105 and CD106 on the surface of P3 cells cultured in vitro completely met the standard.P3 cells were induced to cartilage,fat and bone for 3 weeks.Alcian blue staining showed bright blue cells.After Oil Red O staining,orange red lipid droplets appeared in the cytoplasm.Alizarin red staining showed calcified nodules forming in the center of the aggregated cell masses.These results indicated that SBMSC can differentiate into chondrocytes,adipocytes and osteoblasts after induction and culture in vitro,and have obvious chondrogenic,audiogenic and osteogenic phenotypes,indicating that SBMSC has multidirectional differentiation potential.A complete culture system of SBMSC could be established by adherent culture method to provide seed cells for sheep breeding.