中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (7): 1201-1205.doi: 10.3969/j.issn.1673-8225.2012.07.015

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

胶原酶二步消化法分离培养牛角膜基质成纤维细胞*★

刘曼丽1，邹文进1，黄明汉2，曾  静1   

  1. 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
  • 收稿日期:2011-09-15 修回日期:2011-09-22 出版日期:2012-02-12 发布日期:2012-02-12
  • 通讯作者: 邹文进，副教授，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 bigstone168@163.com
  • 作者简介:刘曼丽★，女，1986年生，广西医科大学在读硕士，主要从事角膜屈光手术、角膜病及眼表疾病学方面的研究。 1084787359@qq.com
  • 基金资助:

    广西壮族自治区自然科学基金资助项目(桂科自0728131)。

Isolation and cultivation of bovine corneal stromal fibroblasts by two-step collagenase digestion method

Liu Man-li1, Zou Wen-jin1, Huang Ming-han2, Zeng Jing1   

  1. 1Department of Ophthalmology, First Affiliated Hospital of Guangxi Medical University, Nanning  530021, Guangxi Zhuang Autonomous Region, China; 2Nanning Aier Eye Hospital, Nanning  530000, Guangxi Zhuang Autonomous Region, China
  • Received:2011-09-15 Revised:2011-09-22 Online:2012-02-12 Published:2012-02-12
  • Contact: Zou Wen-jin, Associate professor, Department of Ophthalmology, First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China bigstone168@163.com
  • About author:Liu Man-li★, Studying for master’s degree, Department of Ophthalmology, First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China 1084787359@qq.com
  • Supported by:

    the Natural Science Foundation of Guangxi Zhuang Autonomous Region, No. 0728131*

PDF

438

被引次数

1

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培养。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学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时间。
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细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，其群体倍增时间为        38.70 h。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。
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
doi:10.3969/j.issn.1673-8225.2012.07.015

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

Abstract:

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.

中图分类号: