人骨髓间充质干细胞的分离培养、多向分化与鉴定

 目的 探索一个高效分离和扩增人骨髓间充质干细胞的方法，并通过形态学和多向分化能力进行鉴定。方法 抽取人胸椎骨髓标本，联合利用密度梯度离心和差异贴壁法分离MSCs，体外扩增后传代，相差显微镜形态学观察和流式细胞仪检测细胞表面标记CD13、CD29、HLA-2、CD34、CD45和HLA-DR。在地塞米松、左旋VitC、b-磷酸甘油、FK506作用下向成骨细胞诱导分化；在TGF-beta 3、重组人胰岛素、丙酮酸钠、亚硒酸等作用下向软骨细胞诱导分化；在地塞米松、IBMX、吲哚美辛的作用下向脂肪细胞诱导分化；在VEGF、bFGF作用下向血管内皮细胞分化。结果 原代和传代细胞呈梭形外观，生长增殖能力良好。细胞表面标记物CD13、CD29，HLA-2阳性，CD34、CD45 和HLA-DR阴性。经定向诱导分化后，细胞分别呈现成骨细胞、软骨细胞、脂肪细胞和血管内皮细胞的表型特征。结论 该方法能从人骨髓中高效分离和扩增MSCs，能成功定向分化为成骨细胞、软骨细胞、脂肪细胞和血管内皮细胞。

Isolation, multiple differentiation and identification of human bone marrow derived mesenchymal stem cells

Objective　To establish a method for effective isolation and multiplication of human bone marrow derived mesenchymal stem cells and verification by morphology and multi-potential differentiation. Methods MSCs were isolated from human bone marrow by combination of gradient centrifugation and different adherent time method. Morphology and growth characteristics were examined by phase contrast microscopy. Cell surface markers CD13, CD29, HLA-2, CD34, CD45 and HLA-DR were tested by flow cytometer. MSCs were induced in vitro respectively to osteoblasts by β-glycerophosphate, L-ascorbicacid-2-phosphate, dexamethasone and FK506; to chondrocytes by TGF-beta 3, recombulin, sodium pyruvate and selenious acid; to adipocytes by IBMX and indomethacin; to endothelial cells by VEGF and bFGF. Results Cells were spindle-shaped and presented active proliferation in primary and passage cultures. The Cell surface markers CD13, CD29, HLA-2 were positive and CD34, CD45 and HLA-DR were negative. Cells were successfully induced into osteoblasts, chondrocytes, adipocytes and endothelial cells. Conclusion Method is established to isolate and multiply MSCs from human bone marrow effectively and to verify by morphology and its multi-potential differentiation.