人骨髓间充质干细胞的分离和体外培养

Isolation and culture in vitro of human bone marrow derived mesenchymal stem cells

目的:分离培养人骨髓间充质干细胞,研究其在体外生长增殖的生物特性。方法:冲洗手术弃骨骨髓,获取骨髓间充质干细胞进行培养,倒置相差显微镜观察细胞生长情况,绘制生长曲线,免疫细胞化学法鉴定细胞表面抗原,进行染色体核型分析。观察冷冻保存细胞复苏后的生长状况。在地塞米松、β-甘油磷酸钠、维生素C的作用下,进行成骨诱导分化。结果:原代和传代培养的细胞呈现梭形外观,具有较强的生长增殖能力;细胞CD44,CD54抗原表达阳性,CD34表达阴性。进行染色体核型分析表明是正常的人二倍体细胞。复苏细胞生物学特性无明显改变。分化细胞表现成骨细胞特性,合成碱性磷酸酶能力增强,矿化结节逐渐出现。结论:建立分离培养人骨髓间充质干细胞和研究其体外培养生物特性的方法,为通过组织工程学方法修复组织损伤奠定基础。

AIM:To culture mesenchymal stem cells(MSCs) derived from human bone marrow, and to study their biological features of growth and proliferation in vitro. METHODS:Human MSCs were washed, obtained and cultured, which derived from deserted bone after operation. The morphology of cells and growth curves of MSCs were studied with inverted phase contrast microscope; the cell surface antigens were detected with immunocytochemical method and chromatosome karyotype analysis.The biological features of MSCs and Cryogentic storaged cells were studied and MSCs differentiated into osteoblasts induced by dexamethasone,β glycerophosphate and ascorbicum. RESULTS:The cultured cells appeared morphologically as spindle shaped and showed active proliferation in vitro in primary and passage cultures; cells were positive for CD44,CD54 and negative for CD34.Chromatosome karyotype analysis showed MSCs were normal human diploid cells.Biological features of cryogentic storaged cells did not alter.Differentiating cells showed the specific property of osteoblast,with the enhancedability of synthesizing alkaline phosphatase(ALP),and the promoted expression of mineralized nodular. CONCLUSIONS:The method is established for culturing human MSCs and studying their biological features in vitro,which provides basis for the amendment of tissue defects by tissue engineering.