人羊膜间充质干细胞储备库的构建

Store bank building for human amniotic mesenchymal stem cells

目的构建人羊膜间充质干细胞库,为间充质干细胞的应用提供充足的细胞来源。方法将胎盘冲洗干净后,分离出羊膜组织,从羊膜组织原代和传代培养羊膜间充质干细胞(hAMSCs)。用MTT法检测细胞生长方式,流式细胞仪分析细胞表型及干细胞标记,使用不同的诱导分化培养基检测鉴定hAMSCs多向分化的能力,细胞周期等鉴定其生物学特性,将磁性纳米颗粒标记的人羊膜间充质干细胞移植到小鼠创面进行细胞功能评价。结果羊膜来源的细胞生长呈典型的成纤维细胞形态。细胞传代过程中未发现细菌、真菌、霉菌、支原体和内毒素等的污染。细胞表达间充质干细胞表面标记CD90和CD105,不表达CD45、CD14、CD34和HLA-DR。在不同的条件培养基培养状态下,细胞可向成骨、成软骨和成脂方向分化。细胞大多数处于G1期,仅少数细胞处于活跃的增殖期G2;建库用人羊膜间充质干细胞可显著促进小鼠创面愈合(P<0.05)。结论所得到的细胞为人羊膜间充质干细胞。羊膜间充质干细胞具有与骨髓间充质干细胞相似的生物学特性。初步建立了库存细胞的生物学特性检测标准和相应的干细胞质量控制体系。

Objective To build human amniotic mesenchymal stem cell bank to provide cells for the application of MSCs. Methods Amniotic membrane was isolated from clean placenta,the human amniotic mesenchymal stem cell underwent primary culture and subculture from amniotic membrane. Cell growth pattern was detected by MTT as- say. Flow eytometry was applied to analyze cell phenotype and stem cell markers. The different differential medium was used to detect its multi-directional differential ability. Results Analysis shows that these amniotic-derived cells are adherent cells with typical fibroblast morphology. Human amniotic mesenchymal stem cells are consistently posi- tive for MSCs labeled with CD90 and CD105, and negative for the hematopoietie markers CD45, CD14, CD34 andHLA-DR. The cells can differentiate into osteoblasts and chondrogenic and adipogenesis under different conditioned mediumcuhured conditions. Cell cycle analysis showed most cells in G1 phase, only a few G2. In vitro experiments show that human amniotic membrane mesenchymal stem cells can promote wound healing in mice （ P 〈 0. 05 ）. Conclusions These cells are human amniotic-derived MSCs. amniotic mesenchymal stem cells have a similar bio- logical characteristics of bone marrow ones.