人羊膜成纤维细胞的分离和鉴定

目的：建立人羊膜成纤维细胞的体外培养方法，通过细胞的形态特征和免疫组织化学进行鉴定，从而获得人羊膜成纤维细胞，为后续的干细胞研究奠定基础。方法：从足月分娩人胎盘剥离羊膜，胰蛋白酶和胶原酶消化后分离人羊膜成纤维细胞，采用含表皮生长因子（EGF）的DMEM培养基进行培养。显微镜下观察细胞形态表现，采用HE染色和免疫组织化学染色分析细胞特征，采用流式细胞术（FCM）分析人羊膜成纤维细胞的纯度。结果：采用胰蛋白酶和胶原酶先后消化获得人羊膜成纤维细胞，显微镜下观察，细胞呈放射状或旋涡状生长。免疫组织化学染色，人羊膜成纤维细胞明显表达成纤维细胞标志物波形蛋白（Vimentin），不同程度地表达S100钙结合蛋白A4（S100A4），不表达上皮细胞标志物角蛋白19（CK19）。流式细胞术分析，人羊膜成纤维细胞的纯度为86.1%。结论：成功建立了人羊膜成纤维细胞分离和鉴定方法，获得的细胞具有成纤维细胞的特异标志和表型特征。

Isolation and identification of human amnion fibroblasts

Objective:To establish the culture method of the human amnion fibroblasts in vitro and to identify based on the cell morphology and immunohistochemistry to get the human amnion fibroblasts,and to lay the foundation for the study on stem cells in the future. Methods:The amnion membrane was removed from the placenta of the parturia,after digestion of trypsin and collagenase,the human amnion fibroblasts were isolated,and DMEM culture including epidermal growth factor(EGF) was used to culture the cells.The morphology of cells was observed by microscope.HE staining and immunohistochemistry were used to analyze the cell characteristics.The purity of human amnion fibroblasts was analyzed by flow cytometry(FCM). Results:After digested with trypsin and collagenase, the human amnion fibroblasts were obtained,and the cells were found in a radial or vortex-like form under microscope.The immunohistochemistry results showed that the human amnion fibroblasts obviously expressed the fibroblast markers Vimentin,expressed the ion binding protein A4 (S100A4) in different degrees,and did not express the epithelial marker keratin CK19.The FCM results showed that the purity of human amnion fibroblasts was 86.1%. Conclusion:The isolation and identification method of human amnion fibroblasts is established successfully,and they have the specific markers and phenotypic characteristics of fibroblasts.