中国组织工程研究2012，Vol.16Issue(7)：1201-1205,5.DOI:10.3969/j.issn.1673-8225.2012.07.015
胶原酶二步消化法分离培养牛角膜基质成纤维细胞
Isolation and cultivation of bovine corneal stromal fibroblasts by two-step collagenase digestion method
摘要
Abstract
BACKGROUND: Obtaining corneal stromal fibroblasts with high purity, activity and biological characteristics closer to in vivostate is the foundation of corneal restoration research.OBJECTIVE: To develop a new culture method for bovine corneal stromal fibroblasts in vitro.METHODS: The bovine corneal stroma was digested by type I collagenase using two-step digestion method to preparesuspension. The cell suspension was transferred into culture bottles for cultivation. Cells in good growth status were subcultured.Cell survival rate was measured by trypan blue staining immediately after digestion. The growth status of bovine corneal stromalfibroblasts was dynamically observed using inverted phase-contrast microscope. Immunohistochemical staining with vimentinwas used to identify the bovine corneal fibroblasts. Cell proliferation was detected using MTT assay. Bovine corneal stromalfibroblasts in logarithmic growth phase were obtained for growth curves and doubling time.RESULTS AND CONCLUSION: Bovine corneal stromal fibroblasts were successfully isolated and cultured in vitro. Microscopicexamination and immunohistochemical staining with vimentin confirmed that the cultured cells were bovine corneal stromalfibroblasts. According to trypan blue staining, the immediate survival rate of bovine corneal stromal fibroblasts was 93.5%. Cellgrowth curve approximated the "S" shape, and cell population doubling time was 38.70 hours. These findings demonstrate thatthe cell culture method of two-step digestion is a simple, economic and efficient method for the primary culture of bovine cornealstromal fibroblasts.关键词
角膜/成纤维细胞/细胞原代培养/细胞形态/生物学特性/消化法分类
医药卫生引用本文复制引用
刘曼丽,邹文进,黄明汉,曾静..胶原酶二步消化法分离培养牛角膜基质成纤维细胞[J].中国组织工程研究,2012,16(7):1201-1205,5.基金项目
广西壮族自治区自然科学基金资助项目(桂科自0728131). （桂科自0728131）
