豚鼠骨髓间充质干细胞的分离培养及成骨诱导

背景：利用骨髓间充质干细胞的取材灵活性及快捷性,对已掌握的培养技术及成骨诱导进一步探索性研究。目的：通过建立豚鼠骨髓间充质干细胞的体外分离培养法,探讨豚鼠骨髓间充质干细胞表型特征以及多项分化潜能。方法：利用贴壁培养法分离纯化豚鼠骨髓间充质干细胞,传代扩增,流式细胞分析检测细胞表面分子CD29、CD44、CD45的表达。分别采用成骨诱导培养液和成脂诱导培养液定向诱导骨髓间充质干细胞向脂肪细胞、成骨细胞分化。结果与结论：原代分离的骨髓间充质干细胞在接种后96 h贴壁,细胞形态为椭圆形,多角形及短梭形,8 d时细胞呈长梭形并达到90%单层融合。经传代扩增,细胞进一步纯化,细胞形态为均一的长梭形并呈漩涡状排列,而且生长速率加快。流式细胞检测 CD29、CD44阳性率分别为95.7%和65.7%。不同诱导剂定向诱导后,经油红O、茜素红S、碱性磷酸酶染色、免疫组织化学Ι型胶原酶鉴定,P3代骨髓间充质干细胞分别向脂肪细胞及成骨细胞分化。结果表明,通过贴壁筛选方法,体外分离培养的豚鼠骨髓间充质干细胞具有很强的增殖能力,并保持稳定的表型特征及多向分化潜能。

Isolation,cultivation and osteogenic induction of guinea pig bone marrow mesenchymal stem cells

BACKGROUND:The culture technique and osteogenic induction of bone marrow mesenchymal stem cells would be further investigated based on flexible and easy harvesting of bone marrow mesenchymal stem cells.OBJECTIVE:To investigate the practical methods of isolation and cultivation of guinea pig bone marrow mesenchymal stromal cells in vitro and to investigate the phenotype characteristics and multilineage differentiation potential of bone marrow mesenchymal stem cells.METHODS:Guinea pig bone marrow mesenchymal stem cells were isolated and purified by adhesion method. After subculturing and proliferation, expression of cell surface molecule CD29, CD44, CD45 was detected by flow cytometry. The multilineage differentiation capability of bone marrow mesenchymal stem cells was examined by culturing cells under conditions favorable for adipogenic and osteogenic differentiation.RESULTS AND CONCLUSION:The primary cultured bone marrow mesenchymal stem cells adhered to plastic surface after 96 hours and exhibited an oval, asteroid or fusiform shape. After 8 days, the cells reached 90 % confluence in a single layer. After subculturing and amplification, the cells were further purified with a uniform firbroblast-like morphology. Flow cytometry analysis showed that the positive rate of CD29 and CD44 was 95.7% and 65.7% respectively. After culture in adipogenic and osteogenic medium respectively, passage 3 cells were successfully induced to differentiate into adipogenic and osteogenic lineages, as identified by oil red O, alizarin red S, alkaline phosphatase staining and immunohistochemical type Ⅰ collagenase staining. These findings suggest that by adherence method, in vitro isolated, cultured guinea pig bone marrow mesenchymal stem cells can greatly proliferate and maintain the phenotypic and functional properties of mesenchymal stem cells.