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

作　　者：刘曼丽[1] 邹文进[1] 黄明汉[2] 曾静[1] 

机构地区：[1]广西医科大学第一附属医院眼科,广西壮族自治区南宁市530021 [2]南宁市爱尔眼科医院,广西壮族自治区南宁市530000

出　　处：《中国组织工程研究》2012年第7期1201-1205,共5页Chinese Journal of Tissue Engineering Research

基　　金：广西壮族自治区自然科学基金资助项目(桂科自0728131)~~

摘　　要：背景:获得纯度高、活性强、生物学特性更接近体内状态的角膜基质成纤维细胞是角膜损伤后修复研究的基础。目的:探索体外培养牛角膜基质成纤维细胞的新方法。方法:用Ⅰ型胶原酶对牛角膜基质层进行二步消化分离后制成细胞悬液转入培养瓶中培养,取生长良好的细胞进行传代养。采用锥虫蓝染色检测消化分离后细胞即刻存活率;倒置相差显微镜动态观察细胞的生长状态;波形蛋白免疫组织化染色鉴定角膜成纤维细胞;MTT法检测细胞增殖情况;取处于对数生长期的细胞,绘制细胞生长曲线并计算细胞群倍增间。结果与结论:在体外成功分离培养牛角膜基质成纤维细胞,显微镜下观察及波形蛋白免疫组织化学染色后证实所培养的胞为角膜基质成纤维细胞。锥虫蓝染色,细胞即刻成活率达93.5%。细胞生长曲线近似"S"形,其群体倍增时间38.70h。说明二步消化法细胞培养技术简便、经济、高效,为原代培养角膜基质成纤维细胞提供了有效渠道。BACKGROUND：Obtaining corneal stromal fibroblasts with high purity,activity and biological characteristics closer to in vivo state 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 Ⅰcollagenase using two-step digestion method to prepare suspension.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 stromal fibroblasts was dynamically observed using inverted phase-contrast microscope.Immunohistochemical staining with vimentin was used to identify the bovine corneal fibroblasts.Cell proliferation was detected using MTT assay.Bovine corneal stromal fibroblasts 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.Microscopic examination and immunohistochemical staining with vimentin confirmed that the cultured cells were bovine corneal stromal fibroblasts.According to trypan blue staining,the immediate survival rate of bovine corneal stromal fibroblasts was 93.5%.Cell growth curve approximated the ＂S＂ shape,and cell population doubling time was 38.70 hours.These findings demonstrate that the cell culture method of two-step digestion is a simple,economic and efficient method for the primary culture of bovine corneal stromal 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 号：R318[医药卫生—生物医学工程]