Abstract: 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.

Key words: immunohistochemistry, flow cytometry, human amnion fibroblasts, HE staining