人脐带间充质干细胞的分离培养及向成骨成脂分化的实验研究

目的 探讨并优化人脐带间充质干细胞(hUCMSCs)体外获取及培养增殖的方法并鉴定;诱导其向成骨细胞和脂肪细胞分化,为其进一步应用奠定基础.方法 用酶消化法分离培养hUCMSCs,通过传代培养、扩增,倒置光学显微镜及原子力显微镜下观察细胞形态;MTT法检测细胞增殖曲线,流式细胞仪检测免疫表型;体外向成骨、脂肪方向诱导分化,并通过特异性染色鉴定其分化能力.结果 采用酶消化法能有效分离纯化hUCMSCs.接种24h,贴壁细胞形态多为长梭型、多边形或成纤维细胞样形态,大小均一.P3、P7代细胞的生长曲线基本一致,增殖能力强.流式细胞仪分析,第3代细胞强烈表达CD29、CD44、CD105,不表达CD34、CD45和HLA-DR.经成骨诱导分化后4周,碱性磷酸酶染色呈强阳性,茜素红染色可见明显钙结节;经成脂诱导3周,油红O染色呈阳性,有明显的脂滴出现.结论 本实验建立了一套体外稳定培养扩增hUCMSCs的方法.所培养的细胞成分单一、扩增迅速、生物学形状稳定,并能使其在体外诱导向成骨细胞和脂肪细胞分化,有望成为组织工程较为理想的种子细胞.

Isolation,culture and identification of mesenchymal stem cells from human umbilical cord as well as their osteogenic and adipogenic differentiation

Objective To establish an optimized method to isolate,culture and identify human umbilical cord mesenchymal stem cells(hUCMSCs) in vitro and induce their osteogenic and adipogenic differentiation.Methods The hUCMSCs were isolated from human umbilical cord by digestion with collagenase.After serial subcultivation in vitro,the stem cells were passaged.Morphologic appearance of hUCMSCs was observed under an optical microscope and atomic force microscope.The proliferation rate was measured by MTT assay.Cell cyc...