新生家兔空肠上皮细胞的体外分离培养与鉴定  

Culture and Identification of Jejunal Epithelial Cells Isolated from Neonatal Rabbits in Vitro

作　　者：范斌[1,2] 余冰[1,2] 田刚[1,2] 张凯[3] 刘汉中[3] 

机构地区：[1]四川农业大学动物营养研究所,成都611130 [2]四川农业大学动物抗病营养教育部重点实验室,成都611130 [3]四川省草原科学研究院,成都611743

出　　处：《动物营养学报》2014年第6期1570-1578,共9页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：四川省科技计划项目"家兔现代产业链关键技术研究与集成示范"(2012NZ0005);四川农业大学"双支计划"项目

摘　　要：本试验旨在建立家兔空肠上皮细胞的体外分离培养方法，为进一步研究外源物质对家兔小肠上皮结构功能的影响及其作用机制提供体外模型。试验以新生新西兰白兔（1~3日龄）空肠为研究对象，利用由链霉蛋白酶E、胶原酶Ⅳ和二硫苏糖醇组成的混合消化液消化空肠组织，通过2%山梨醇密度梯度离心来富集隐窝细胞团。运用相差消化法和差速贴壁法纯化家兔空肠上皮细胞，并从形态学观察、碱性磷酸酶染色、细胞角蛋白8免疫荧光染色和肠上皮细胞标志物基因表达分析4个方面鉴定家兔空肠上皮细胞。结果表明：应用链霉蛋白酶E、胶原酶Ⅳ和二硫苏糖醇混合消化液可获得大量的细胞团，细胞团12 h贴壁并向周围辐射生长，生长的细胞呈典型的三角形或多角形，5~7 d融合成片，呈单层生长、互不重叠；原代细胞能够进行传代、纯化；经碱性磷酸酶染色和细胞角蛋白8免疫荧光染色均呈阳性；可表达肠上皮细胞标志蛋白（脂肪酸结合蛋白2、肠肽酶、E-钙黏蛋白、紧密连接蛋白-4）的基因。以上结果表明，本试验成功建立了家兔空肠上皮细胞的体外分离培养方法。This study was aimed to establish a method to culture jejunal epithelial cells （JEC） isolated from rabbits, and provide an in vitro model for the further study of the effects of exogenous substances on rabbit intestinal epithelial structure and mechanism. Newborn New Zealand white rabbits （1 to 3 days of age） were used, jejunum was removed and JEC were isolated by the application of digestive juice mixed with pronase E, collagenase Ⅳ and dithiothreitol （DTT）, and the crypt cell aggregates were enriched by density gradient centrifugation of 2% sorbitol. The rabbit JEC were purified by differential digestion method and differential velocity adherent method. Cells were identified by morphological observation, alkaline phosphatase staining, cytokerain-8 immunofluorescence staining and the gene expression analyses of enterocyte markers. The results showed that the application of digestive juice mixed with pronase E, collagenase Ⅳ and DTT could successfully get a lot of rabbit JEC aggregates, which readily adhered to culture dishes within 12 hours and grown to around radially, growing cells showed typical triangular or polygonal, reached confluence within 5 to 7 days, and showed monolayer and non-overlapping; cells could be passaged and purified; cells were stained positive for alkaline phosphatas staining and cytokerain-8 immunofluorescence staining; enterocyte markers, such as fatty acid binding protein 2, enterokinase, E-cadherin, claudin-4, were expressed in JEC. These results show that an ideal method for isolating and culturing epithelial cells of rabbit jejunum is established.

关 键 词：新西兰白兔 空肠上皮细胞 原代培养 鉴定 

分 类 号：Q813.1[生物学—生物工程]