猪支气管黏膜上皮细胞的分离培养和传代研究  

Research of Dissociation Culture and Subculture of Epithelium Cells from Swine Bronchia

作　　者：孙裴[1] 张彦明[1] 杨小云[1] 温元鹏[1] 张浩[1] 何建锋[1] 郭抗抗[1] 党如意[1] 

机构地区：[1]西北农林科技大学动物医学院,杨凌712100

出　　处：《畜牧兽医学报》2008年第6期771-777,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(30471290)

摘　　要：利用气管-支气管扎结灌注酶冷消化法分离气管黏膜上皮细胞,比较胰蛋白酶和链霉蛋白酶消化效果,并研究含不同细胞生长因子和血清浓度的培养体系来寻找经济、易重复的气管上皮细胞最适培养技术。用抗8/18细胞角蛋白免疫组化法鉴定气管上皮细胞,用流式细胞仪检测细胞凋亡来分析本研究中气管上皮细胞最大传代次数。研究结果表明胰蛋白酶消化气管所得的目的细胞量小、活性差、细胞贴壁和生长困难,链霉蛋白酶灌注冷消化法可获得数量大、活性高、纯度好的目的细胞;基础的上皮细胞培养体系中含低浓度血清、转铁蛋白和人表皮生长因子可以有效保证气管上皮原代和传代生长。本研究所分离的气管上皮细胞可连续传到第6代,传代细胞活性好、纯度高,可为进一步的气管上皮细胞永生化研究提供材料和奠定技术基础。By the method of douche, trachea-bronchia epithelium was digested overnight and separated. The digestion products of the two enzymes, Trypsin and Pronase, were compared. Different cell culture mediums which including different cell factors and serum concentration were set up in order to select optimal tracheabronchia epithelium culture technology which is economical and easy to be repeated. Through immunohistochemistry of cell No. 8/18 keratins and testing cell program death with flow cytometer, the maximal number of possible cell generation was measured. The research results indicated that by the method of Trypsin digestion only a small quantity of cells were gotten and the acquired cells were very difficult to conglutinate and grow, and by Pronase digestion a number of cells were obtained which is pure and highly active. To the system of basic trachea-bronchia epithelium culture the use of low serum concentration, transferrin and epidermal growth factor are enough to assure primary trachea-bronchia epithelium and its subculture to grow well. The trachea-bronchia epithelium separated by us could be successively propagated six times and keep active well and pure highly, which may provide ideal research material and basis on technology for study of immortalisation of swine trachea-bronchia epithelium.

关 键 词：分离培养 气管-支气管 上皮细胞 传代 

分 类 号：S858.28[农业科学—临床兽医学] Q959.836[农业科学—兽医学]