解剖学杂志2024，Vol.47Issue(5)：385-388,4.DOI:10.3969/j.issn.1001-1633.2024.05.003
大鼠肺成纤维细胞原代分离培养方法优化
Optimization of primary isolation and culture methods for rat pulmonary fibroblasts
摘要
Abstract
Objective:To explore a rapid isolation and culture method for rat pulmonary fibroblasts(PFs),providing more scientific and convenient technical support for in vitro studies of pulmonary fibrosis.Methods:Lung tissues from newborn SD rats were dissected.Type Ⅱ collagenase/pancreatin mixed enzyme digestion method was used.Microscopic observation and analysis were conducted on the cell suspension and precipitate after each tissue digestion.The cell suspension was selectively collected,and the differential adhesion time of cells was gradient grouped to optimize the best cell adhesion time point.Coomassie brilliant blue staining was used to observe the uniformity of cell morphology.Vimentin labeling was used to identify the purity of PFs,and α-SMA labeling was used to identify the differentiation ability of cells.Cell growth curve was plotted to detect cell growth activity.Results:After the first two 8-minute digestions,the cell suspension mainly contained blood cell components,and the precipitate consisted mostly of blood clots and substantial tissue components.After the 3rd-6th digestion,the cell suspension mainly contained nucleated cell components,and the digestion solution precipitate consisted mostly of collagen,adhesive proteins,and other matrix clumps.The cell suspension from the 3rd-6th digestion was selected for differential adhesion separation of cells.Comprehensive analysis of cell quantity and purity indicated that a 35-minute differential adhesion time was optimal,resulting in high cell viability.Cells reached logarithmic growth phase within 24 hours across the first to fourth generations.Conclusion:Sequential enzyme digestion,selective collection of cell suspensions,and a 35-minute differential adhesion separation significantly improve the isolation of PFs.关键词
肺/纤维化/成纤维细胞/细胞培养/大鼠Key words
lung/fibrosis/fibroblast/cell culture/rat分类
医药卫生引用本文复制引用
柏梦如,徐佩雯,巩赫喆,车继超,王丹,田香勤..大鼠肺成纤维细胞原代分离培养方法优化[J].解剖学杂志,2024,47(5):385-388,4.基金项目
河南省科技攻关项目(232102311072) （232102311072）
