确证家兔外周血中存在具有多向分化能力的间充质干细胞

本研究确证家兔外周血中存在具有多向分化潜能的间充质干细胞（MSC）。取成年新西兰大白兔，经每日皮下注射粒细胞集落刺激因子30μg/kg动员6d后采集外周血样品，用Ficoll密度梯度离心和贴壁培养法分离纯化外周血间充质干细胞（PBMSC），镜下观察其生长形态并绘制增殖曲线；流式细胞术分析其表型特征；并分别对培养的PBMSC进行成骨、成软骨及成脂细胞诱导分化能力鉴定。结果表明：①原代培养24h后，可见较多短梭形及多角形贴壁细胞，3-4d后出现集落样生长，传代培养的PBMSC形态均一、呈长梭形漩涡状生长；②PBMSC增殖能力旺盛，培养2d后进入对数生长期，细胞倍增时间为37．4h；③流式细胞术检测显示，培养的PBMSC高表达CD29，不表达CD14；④PBMSC经成骨诱导培养21d后，茜素红染色显示有明显的钙结节形成；经微团无血清软骨培养体系诱导培养21d后，细胞团切片，阿利新蓝染色呈强阳性反应；经成脂诱导培养21d后，油红染色显示诱导细胞胞浆内有大量脂滴。结论：成功从兔外周血中分离培养了PBMSC，在适宜诱导培养条件下具有向成骨、成软骨、成脂分化的能力。

Verification of Multipotential Mesenchymal Stem Cell Presence in Peripheral Blood of Rabbits

The aim of this study was to verify the presence of multipotential mesenchymal stem cells in peripheral blood （PBMSC） of rabbits. For mobilization, granulocyte-colony-stimulating factor 30 μg/（ kg ·d） was injected into New Zealand White rabbits subcutaneously for 6 d, then the PBMSC were isolated from peripheral blood of rabbits by density gradient centrifugation and adhesive culture. The morphology of cell proliferation was observed by microscopy, the proliferative curve of cells was drawn. The phenotypes of PBMSC were detected by flow cytometry, the differential caparitity of PBMSC into osteocytes, chrondrocytes and adipocytes was identified. The results showed that the morphology of subcultured PBMSC were spindle or polygonal shaped, and cell population doubling time was 37.4 h. The isolated PBMSC expressed mesenchymal marker CD29, but not expressed hematopoietic marker CD14. Under specific induction conditions, PBMSC demonstrated multipotency to differentiate into osteocytes, chondrocytes and adipocytes. It is concluded that PBMSC are successfully isolated from peripheral blood and cultured, and their multipotential capability of differentiation into osteocytes, chrondrocytes and adipocytes are verifyed.