树鼩小肠上皮细胞的体外分离培养与鉴定  

Culture and Identification of Intestinal Epithelial Cells Isolated from Tree Shrew in vitro

作　　者：尹博文[1] 陈玲霞[1] 李振宇[1] 李晓飞[1] 宋庆凯 苗雨润[1] 孙晓梅[1] 

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所树鼩种质资源中心云南省重大传染病疫苗研发重点实验室,昆明650118

出　　处：《实验动物科学》2017年第2期11-15,共5页Laboratory Animal Science

基　　金：国家科技支撑计划(No.2014BAI01B00);云南省联合支持国家计划项目(No.2015GA009)

摘　　要：目的从树鼩小肠分离小肠上皮细胞,探索分离培养方法和条件,为药物开发和病原感染机制研究提供体外细胞模型,并且为建立树鼩小肠上皮细胞系奠定基础。方法采用胶原酶Ⅺ和中性蛋白酶Ⅰ及DTT联合消化树鼩小肠组织块,得到的细胞经完全培养液培养,用相差显微镜观察细胞形态,通过MTT法测定细胞生长曲线,并采用细胞角蛋白荧光染色等方法鉴定细胞。结果经优化分离法得到的肠上皮隐窝细胞团在24 h后贴壁,6 d汇合成片,在显微镜下观察发现细胞团延辐射状向外长出铺路石样和多角状的细胞;第二代后,每隔2~3 d可传代1次;细胞接种3~6 d为对数生长期;细胞角蛋白18进行免疫荧光鉴定后确定为小肠上皮细胞。结论本实验成功地从树鼩小肠分离到了小肠上皮细胞,并且建立了树鼩小肠上皮细胞体外培养的方法。Objective To isolate intestinal epithelial eells（lECs） from the tree shrew, to establish a cellular model for drug mechanism research, and to lay the foundation for establishment of intestinal epithelial cell lines. Method Collagenase type XI, and neutral protease type I and DTT were used for digestion of tree shrew small intestine. Cells morphology was observed by phase contrast microscopy, identified by cytokeratin Immunofluorescenee assay. Result Isolated intestinal epithelial crypts attachment were performed in 24 h, confluent in 6 d. Epithelial outgrowth from isolated crypts were formed with confluent monolayers cobblestone and multi-angular morphology. Cells were passaged every 2 ~ 3 d from the nest generation, and between the third and sixth day from the day when cells were plated the cells got into the logarithmic growth phase. The cells were stained positive for eytokeratin immunofluoreseence staining. Conclusion Intestinal epithelial ceils were sueeessfully isolated from the tree shrew, and the method of isolation for tree shrew＇ s intestinal epithelial cells was established.

关 键 词：树鼩 小肠上皮细胞 酶消化 免疫荧光 鉴定 

分 类 号：Q813.11[生物学—生物工程]