犬骨髓间充质干细胞分离培养及鉴定

Culture and Identification of Marrow Mesenchymal Stem Cells in Dog

目的建立犬髂骨骨髓间充质干细胞分离、培养的方法,并对间充质干细胞进行鉴定。方法采用密度梯度分离和贴壁培养相结合的方法分离培养犬的骨髓间充质干细胞(MSCs),并诱导间充质干细胞向成骨细胞、成脂肪细胞方向分化。相差显微镜观察其形态学变化,组化染色测定其分化结果。结果原代培养4～5d,镜下见细胞呈梭形或多角形,有较多的突起,胞核明显呈卵圆形,可见1-3个核仁,胞质丰富。成骨方向诱导形成的矿化结节茜素红染色阳性,碱性磷酸酶(alkaline phosphatase,ALP)染色阳性;成脂方向诱导形成的脂肪细胞苏丹Ⅳ染色阳性。结论采用密度梯度分离和贴壁培养相结合的方法能够成功分离和培养犬的骨髓间充质干细胞。

Objective To establish a method for isolation, culture and differentiation of bone marrow mesenchymal stem cells （MSCs） from dog ilium. Methods MSCs were isolated from dog ilium bone marrow and cultured and induced their differentiation into osteoblasts and adipocytes which were identified by histochemistry staining. Morphological changes were observed by phase contrast microscope. Results After seeded 4-5 days, the cultured adherent cells showed spindle and polygonal shape ,with many tentacles and obvious nucleus of orbicular-ovate shape and 1-3 nucleoli and abundant cytoplasm.The osteoblasts which synthesized mineralization node were seen by Alizarin red staining and by alkaline phosphatase staining.The differentiated lipid droplets were observed by Sudan IV. Conclusion MSCs were isolated and cultured successfully from dog ilium bone marrow by combination of gradient centrifugation and adherent wall culture.