人脐带间充质干细胞的分离培养及多向分化潜能研究

Research on isolation,culture and multiple differentiation potential of human umbilical cord mesenchymal stem cells

建立人脐带间充质干细胞的分离培养方法,并鉴定其生物学特性及多向分化潜能。采集健康产妇足月剖宫产中的胎儿脐带组织,通过组织块贴壁法进行原代培养,分别采用流式细胞仪和RT-PCR方法检测细胞表面标志物和多能性相关基因的表达,通过向成脂、成骨、成软骨分化,鉴定其多向分化潜能。结果显示:脐带组织贴壁培养14 d,可见组织块周围出现大量贴壁生长的梭形样细胞,获得的细胞高表达间充质干细胞表面标志物CD44、CD105,低表达造血细胞表面标志物CD34、CD45;同时表达多能性相关基因Oct4和Nanog,经组织染色和RT-PCR鉴定,其具有向成脂、成骨和成软骨分化的潜能。人脐带间充质干细胞可在体外扩增培养,表达多能性相关基因,并具有多向分化潜能,可作为种子细胞用于组织工程。

The method of isolation and culture of human umbilical cord mesenchymal stem cells(hUCMSCs)was established,and their biological characteristics and multi-differentiation potential were identified.Umbilical cord tissues from healthy maternal in full-term cesarean section were collected and primary culture was performed by tissue explants adherent method.The expression of cell surface markers and the pluripotent related genes were detected by flow cytometry and RT-PCR,respectively.Multi-differentiation capacity was identified by adipogenic,osteogenic and chondrogenic differentiation.Results showed that a lot of cells were appeared around the umbilical cord tissue piece on the 14 th day,the cells highly expressed mesenchymal stem cell surface markers CD44 and CD105,but negative for hematopoietic cell surface markers CD34 and CD45.Moreover,the cells also expressed pluripotent related genes Oct4 and Nanog.The cells potential to differentiate into adipocytes,osteocytesand chondrocytes was identified by tissue staining and RT-PCR.In conclusion,hUCMSCs can be proliferated and cultured in vitro,expressed pluripotent related genes.And they had the multi-directional differentiation potentiality and could be used as seed cells for tissue engineering.