新生仔猪口腔黏膜上皮细胞的体外培养方法  

The Culturing Method of Neonatal Piglet Oral Mucosal Epithelial Cells in vitro

作　　者：马瑞丽[1,2] 张彦明[1] 崔红杰[1] 郭抗抗[1] 

机构地区：[1]西北农林科技大学动物医学院,杨凌712100 [2]西北农林科技大学生命科学学院,杨凌712100

出　　处：《畜牧兽医学报》2013年第9期1445-1453,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：陕西省13115科技创新工程重大科技专项(2010ZDKG-71)

摘　　要：为了建立仔猪口腔黏膜上皮细胞（oral mucosal epithelial cells，OMEC）的体外培养方法，取新生仔猪的口腔颊膜组织，分别采用组织块培养法、胰蛋白酶消化法、DispaseⅡ和胰蛋白酶联合消化培养法、DispaseⅡ处理联合组织块培养法，对口腔黏膜上皮细胞进行原代和传代培养，用倒置显微镜观察培养细胞形态学特征及生长情况，对传代培养的OMEC，采用间接免疫荧光法检测广谱细胞角蛋白的表达情况，并进行透射电镜观察，用MTT法测定细胞的生长曲线，流式细胞仪检测其细胞周期。结果表明，在采用相同培养基条件下，采用组织块培养法、胰蛋白酶消化法不能成功培养仔猪OMEC，用DispaseⅡ和胰蛋白酶联合消化法培养的细胞不易贴壁，培养困难，采用DispaseⅡ处理联合组织块培养法培养的细胞增殖能力强，细胞纯度高，可连续传到10～11代，经鉴定具有OMEC特征。本试验成功建立了仔猪口腔黏膜上皮细胞的原代及传代培养方法，为进一步研究OMEC的永生化及猪口蹄疫致病机理和疫苗提供理想实验材料。The aim of this study was to establish the culture method of piglet oral mucosal epithe- lial cells in vitro. Piglet buccal cells were cultured and subcultured with the direct explant tech- nique, trypsin digestion, combined method of Dispase Ⅱ and trypsin digestion, and Dispase Ⅱ combined with explant technique. Morphological characteristics were observed under the light microscope and transmission electron microscope （TEM）. For second cultures, the expression of cytokeratin was detected by indirect immunofluorescence, cell growth curve was measured by MTT method, cell cycle distribution was evaluated by flow cytometry. The results showed that in the same culture medium the explant technique and enzymatic method were not successful, the cells obtained by the method of combination of dispase Ⅱ and trypsin digestion were difficult to at- tach and grow, but the cells obtained by the combination of Dispase Ⅱ and explant technique pro- liferated quickly, and were of high purity, which can be passaged continuously till to the 10-11 generations, and the cells were identified with the characteristics of oral mucosal epithelial cells. The culture method of piglet oral mucosal epithelial cells in vitro has been established successful- ly, which may provide ideal research material for study the immortalization of oral mucosal epi- thelial cells, swine foot-and-mouth disease （FMD） pathogenesis and its vaccine.

关 键 词：口腔黏膜上皮细胞 体外培养 DispaseⅡ联合组织块培养法 新生仔猪 

分 类 号：Q813.1[生物学—生物工程]